Methods and compounds for prevention and treatment of elevated intraocular pressure and related conditions

ABSTRACT

A GPCR-like protein is described, as well as inhibitory/antagonistic compounds and compositions comprising such inhibitors/antagonists of the protein. Such compounds may be used for treating elevated intraocular pressure and conditions associated with elevated intraocular pressure, such as glaucoma.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. provisional application No.60/367,513 filed Mar. 27, 2002.

FIELD OF THE INVENTION

The invention relates to methods and compounds for the prevention and/ortreatment of ocular hypertension or elevated intraocular pressure, andrelated conditions such as glaucoma.

BACKGROUND OF THE INVENTION Glaucoma and Intraocular Pressure

Glaucoma is characterized by optic nerve head excavation which can leadto loss of peripheral vision and sometimes loss of central vision.Glaucoma is the second leading cause of vision loss worldwide; anestimated 66.8 million people will have primary glaucoma and 6.7 millionwill be bilaterally blind due to this disorder. In the United States,glaucoma is the second leading cause of permanent blindness and theleading cause among African Americans. While glaucoma is treatable andvision loss can be prevented, once vision loss occurs it isirreversible. Elevated intraocular pressure (IOP) is a major risk factorfor the development of glaucoma, but it is not found in all patientswith the disease (Sommer, A. et al., Arch. Ophthalmol. 109: 1090-1095(1991)). Glaucoma associated with elevated IOP is divided into threemajor categories: open angle, closed angle and developmental. Each ofthese categories is further divided into primary and secondary forms,and by the age of onset. Drugs that lower IOP, such as latanoprost,unoprost, timolol which target prostaglandin F_(2α) and β₂ adrenergicreceptors are being currently used to prevent and delay oculardegeneration in glaucomatous patients.

There is therefore a continued need to identify and characterize targetsand in turn products for the prevention and treatment of oculardisorders, such as glaucoma.

SUMMARY OF THE INVENTION

The invention relates to R-14 nucleic acids and polypeptides andcompounds capable of lowering intraocular pressure and uses thereof.

Accordingly, in an aspect, the invention provides a substantially purepeptide compound of Formula I:Z₁-X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-Z₂  Iwherein:

-   X₁ is selected from the group consisting of Phe, His, Ile and    related amino acids;-   X₂ is selected from the group consisting of Ser, Ile, Phe and    related amino acids-   X₃ is selected from the group consisting of Leu, Ile, Asp and    related amino acids-   X₄ is selected from the group consisting of Thr, Cys, Ser and    related amino acids possessing side chains containing sulfhydryl,    hydroxyl or H-bond forming groups;-   X₅ is selected from the group consisting of Gln, Ser, Thr and    related amino acids;-   X₆ is selected from the group consisting of Lys, Pro, Glu and    related amino acids;-   X₇ is selected from the group consisting of Tyr, Leu, Cys and    related amino acids;-   X₈ is selected from the group consisting of Cys, Arg, Trp and    related amino acids;-   Z₁ is an N-terminal group of the formula H₂N—, RHN— or, RRN—;-   Z₂ is a C-terminal group of the formula —C(O)OH, —C(O)R, —C(O)OR,    —C(O)NHR, —C(O)NRR;-   R at each occurrence is independently selected from (C₁-C₆) alkyl,    (C₁-C₆) alkenyl, (C₁-C₆) alkynyl, substituted (C₁-C₆) alkyl,    substituted (C₁-C₆) alkenyl, or substituted (C₁-C₆) alkynyl; and-   “—” is a covalent linkage.

The invention further provides a substantially pure synthetic peptidecompound or recombinant peptide compound, said compound having a domainof Formula II:-X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈  IIwherein X₁, X₂, X₃, X₄, X₅, X₆, X₇, X₈ and “—” are as defined above.

In embodiments, the compound is selected from the group consisting of:FSLTQKYC; HIICSPLR; and IFDSTECW. In embodiments, the domain is selectedfrom the group consisting of: FSLTQKYC; HIICSPLR; and IFDSTECW.

The invention further provides a method of lowering intraocular pressurein a subject, said method comprising administering to said subject aneffective amount of the above-mentioned compound.

The invention further provides a method of treating in a subject acondition associated with elevated intraocular pressure, said methodcomprising administering to said subject an effective amount of theabove-mentioned compound.

In embodiments, the methods comprise administering said compound to aneye of said subject.

The invention further provides a pharmaceutical composition comprisingthe above-mentioned compound in admixture with a pharmaceuticallyacceptable carrier. In an embodiment, the composition is formulated foradministration to an eye of a subject.

The invention further provides a commercial package comprising theabove-mentioned compound together with instructions for its use. Inembodiments, the use is selected from the group consisting of: (a)lowering intraocular pressure in a subject; and (b) treating a conditionassociated with elevated intraocular pressure.

The invention further provides an isolated nucleic acid comprising asequence that encodes the above-mentioned domain. The invention furtherprovides a vector comprising the nucleic acid operably-linked to atranscriptional regulatory element. The invention further provides ahost cell comprising the vector.

The invention further provides a method of producing the above-mentionedpeptide compound, comprising culturing the above-mentioned host cellunder conditions permitting expression of the peptide compound.

The invention further provides an isolated nucleic acid comprising asequence that encodes a polypeptide comprising at least 278 amino acidsof SEQ ID NO:2. In an embodiment, the polypeptide comprises SEQ ID NO:2.In an embodiment, the nucleic acid comprises SEQ ID NO:1 or a sequencesubstantially identical thereto.

The invention further provides a substantially pure polypeptidecomprising at least 278, consecutive amino acids of SEQ ID NO:2. In afurther embodiment, the polypeptide comprises SEQ ID NO:2.

The invention further provides a vector comprising the above-mentionednucleic acid operably linked to a transcriptional regulatory element.The invention further provides a host cell comprising theabove-mentioned vector.

The invention further provides a method of producing the above-mentionedpolypeptide, comprising culturing the above-mentioned host cell underconditions permitting the expression of the polypeptide.

The invention further provides a method for identifying a compound for:(i) lowering intraocular pressure in a subject; (ii) treating acondition associated with elevated intraocular pressure; or (iii) both(i) and (ii); said method comprising: (a) providing a cell expressing anR-14; (b) contacting the cell with a test compound; and determiningwhether R-14 activity is decreased in the presence of said testcompound, said decrease in activity being an indication that saidcompound may be useful for: (i) lowering intraocular pressure in asubject; (ii) treating a condition associated with elevated intraocularpressure; or (iii) both (i) and (ii). In an embodiment, said decrease inactivity is an indication that said compound is an R-14 antagonist. Inan embodiment, R-14 comprises a polypeptide comprising SEQ ID NO:2, anactive fragment thereof, or a sequence substantially identical thereto.

The invention further provides a use of the above-mentioned compoundfor: (a) lowering intraocular pressure in a subject; (b) treating acondition associated with elevated intraocular pressure; or (c) both (a)and (b).

The invention further provides a use of the above-mentioned compound orthe above-mentioned composition for the preparation of a medicament. Inan embodiment, the medicament is for: (a) lowering intraocular pressurein a subject; (b) treating a condition associated with elevatedintraocular pressure; or (c) both (a) and (b).

In an embodiment, the above-mentioned condition is glaucoma.

In an embodiment, the above-mentioned subject is a mammal, in a furtherembodiment, a human.

In further embodiments, Z₁ is selected from the group consisting of aproton, a sequence of 1-3 amino acids, or a blocking group such as acarbamate group, an acyl group composed of a hydrophobic moiety such ascyclohexyl, phenyl, benzyl, short chain linear and branched alkyl groupsof 1-8 carbons. In further embodiments Z₂ is selected from the groupconsisting of proton, NH₂, 1-3 amino acids as well as arylalkyl aminessuch as benzylamine, phenylethylamine, phenylpropylamine, and aliphaticamines possessing short chain linear and branched alkyl groups of 1 to 8carbons.

In another aspect, the invention provides a method for formulating amedicament, the method comprising admixing a compound of the inventionwith a pharmaceutically acceptable carrier.

In another aspect, the invention provides a method of loweringintraocular pressure in a subject, the method comprising inhibitingexpression and/or activity of an R-14 protein in the subject. In anembodiment, the R-14 protein comprises a polypeptide selected from thegroup consisting of:

-   (a) the polypeptide of SEQ ID. NO. 2; and-   (b) a polypeptide encoded by a first nucleic acid that is    substantially identical to a second nucleic acid capable of encoding    the polypeptide of SEQ ID NO. 2. In an embodiment, the second    nucleic acid is as set forth in SEQ ID NO. 1.

In an embodiment, the above-noted method comprises administering to thesubject an effective amount of an agent capable of modulating R-14activity. In an embodiment, the agent is a compound of the invention. Ina further embodiment, the method comprises administering to the subjecta therapeutically-effective dose of the above-mentioned composition. Inan embodiment, the dose is of about 0.001 mg to of about 100 mg.

The invention further provides a method of lowering intraocular pressurein a subject, the method comprising administering to the subject aneffective amount of a compound of the invention. In an embodiment, themethod comprises administering to the subject atherapeutically-effective dose of the above-mentioned composition. In anembodiment, the dose is of about 0.001 mg to of about 100 mg.

The invention further provides a method of preventing or treating in asubject a condition associated with elevated intraocular pressure, themethod comprising inhibiting expression and/or activity of an R-14protein in the subject. In an embodiment, the R-14 protein comprises apolypeptide selected from the group consisting of:

-   (a) the polypeptide of SEQ ID NO. 2; and-   (b) a polypeptide encoded by a first nucleic acid that is    substantially identical to a second nucleic acid capable of encoding    the polypeptide of SEQ ID NO. 2. In an embodiment, the second    nucleic acid is as set forth in SEQ ID NO. 1.

In an embodiment, the method comprises administering to the subject aneffective amount of an agent capable of modulating R-14 activity. In anembodiment, the agent is a compound of the invention. In a furtherembodiment, the method comprises administering to the subject atherapeutically-effective dose of the above-mentioned composition. In anembodiment, the dose is of about 0.001 mg to of about 100 mg.

The invention further provides a method of preventing or treating in asubject a condition associated with elevated intraocular pressure, themethod comprising administering to the subject an effective amount of acompound of the invention. In a further embodiment, the method comprisesadministering to the subject a therapeutically-effective dose of theabove-mentioned composition. In an embodiment, the dose is of about0.001 mg to of about 100 mg.

The invention further provides a commercial package comprising an agentcapable of modulating R-14 activity together with instructions for:

(a) lowering intraocular pressure in a subject;

(b) preventing or treating a condition associated with elevatedintraocular pressure; or

(c) both (a) and (b).

In an embodiment, the R-14 protein comprises a polypeptide selected fromthe group consisting of:

(i) the polypeptide of SEQ ID NO. 2; and

(ii) a polypeptide encoded by a first nucleic acid that is substantiallyidentical to a second nucleic acid capable of encoding the polypeptideof SEQ ID NO. 2.

The invention further provides a method for identifying and/orcharacterizing a compound for lowering intraocular pressure, the methodcomprising assaying the activity of an R-14 in the presence of a testcompound, to identify a compound that acts as an R-14 antagonist,wherein antagonist activity is indicative that the test compound may beuseful for lowering intraocular pressure.

The invention further provides a method for identifying and/orcharacterizing a compound for lowering intraocular pressure, the methodcomprising:

-   (a) contacting a test compound with a host cell expressing a    polypeptide selected from the group consisting of:-   (i) the polypeptide of SEQ ID NO. 2; and-   (ii) a polypeptide encoded by a first nucleic acid that is    substantially identical to a second nucleic acid capable of encoding    the polypeptide of SEQ ID NO. 2; and-   (b) assaying activity of an R-14 in the presence of the test    compound, to identify a compound that acts as an R-14 antagonist,    wherein antagonist activity is indicative that the test compound may    be useful for lowering intraocular pressure. In an embodiment, the    compound may be used for the prevention and/or treatment of a    condition associated with intraocular pressure.

In an embodiment, the above-mentioned subject is a mammal, in a furtherembodiment, a human.

In an embodiment, the above-mentioned condition is glaucoma.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: (A) Agarose gel electrophoretic resolution of ˜960 bp product(arrow) obtained by PCR amplification as described in Example 1. −Bac:No plasmid DNA; +Bac: contains plasmid DNA (0.1 μg); MW std: λ Hind IIIdigest. (B). The nucleotide sequence of the R-14 reading frame (SEQ IDNO:1). (C) R-14 polypeptide sequence (SEQ ID NO:2).

FIG. 2: Agarose gel electrophoretic analysis of RT-PCR products foranalysis of R-14 expression as described in Example 2. Iris: samplederived from Iris tissue; HTM: sample derived from human trabecularmeshwork tissue.

FIG. 3: Immunoblot analysis of expression of cloned human R-14 receptorin human cells as described in Example 3. Arrow shows R-14immunoreactive band.

FIG. 4: Graphical results of identification and characterization ofinhibitors of R-14 protein as described in Example 4.

FIG. 5: Graphical results of comparison of the efficacy of peptide 1405with Latanoprost and Timolol as described in Example 5.

FIG. 6: Graphical results of analysis of selectivity of peptides 1402and 1405 as described in Example 6.

FIG. 7: Graphical results of effect of peptides 1402 (A) and 1405 (B) onintraocular pressure in rabbits, as described in Example 7.

Other objects, advantages and features of the present invention willbecome more apparent upon reading of the following non-restrictivedescription of preferred embodiments with reference to the accompanyingdrawings, which is exemplary and should not be interpreted as limitingthe scope of the present invention.

DETAILED DESCRIPTION OF THE INVENTION Identification of Novel GPCRs

Use of low stringency hybridization, differential display, microarrays,subtractive hybridization and other techniques followed by cloning ofdifferentially expressed genes in tissues have disclosed several newcDNAs which could potentially code for new G protein-coupled receptors.Following the elucidation of the draft sequence of the human genome(Venter, J C. et al., Science. 291: 1304-1351 (2001); InternationalHuman Genome Sequencing Consortium. Nature. 409: 860-921 (2001)), manyGPCR-like sequences have been identified based on their canonical seventransmembrane topology as well as conserved protein motifs (Howard, D Aet al., 2001. Trends Pharmacol. Sci. 22(3): 132-140). These uniqueGPCR-like sequences number ˜1000 of which nonolfactory GPCR sequencesare estimated to be ˜616.

Gonzalez et al. (Gonzalez, P. et al., 2000. Invest. Ophthalmol. Vis.Sci. 41: 3678-3673), carried out single pass sequencing of 1060 cDNAclones isolated from human trabecular meshwork, including a sequencehaving homology to MAS-related G-protein coupled receptor (denoted asHTMI-0025, and corresponding to sequences Hs11_(—)9464 and Hs11_(—)24438found in the human chromosome 11 working draft sequence).

Dong et al. (Dong, X. et al. 2001. Cell 106:619-632) cloned severalmas-related cDNAs from mouse embryos lacking neurogenin gene, withsubsequent searching among human genome sequences revealing four relatedgenes (called Hs_mrgX1-4).

When the physiological ligand(s) of a GPCR is not known, then it istermed an orphan GPCR. In order to find ligands of these orphan GCPRs,random libraries containing natural peptides isolated from the tissuesor those containing small molecules produced by combinatorial chemistryor from natural sources are screened in cell-based and in vitro assays.Selective modulators of the orphan GPCR can be used to assess itsfunction in physiology and pathophysiology using appropriate animalmodels. Finding a function of the orphan GPCR is a first step towardsanalyzing its potential as a new drug target.

The studies described herein relate to a novel GPCR protein expressed intrabecular meshwork, nucleic acids capable of encoding it, and peptidecompounds capable of modulating phenomena in ocular tissue, notablyintraocular pressure.

In one aspect, the invention provides an isolated a GPCR-like readingframe, which is capable of expressing mRNA and protein, and isassociated with a role in ocular outflow in animals. The inventionfurther provides three antagonists of the GPCR which could be used forlowering intraocular pressure, and thus for prevention/treatment ofdisorders associated with elevated intraocular pressure, such asglaucoma and related conditions.

As described herein, applicants have cloned a genomic DNA fragment of0.97 Kb in length from a BAC clone and have shown that it contained asingle exon encoding a 322 amino acid long protein. This protein istermed as R-14. From in silico analysis, R-14 appears to be an integralmembrane protein containing seven prominent transmembrane domains andmany protein motifs of G protein-coupled receptors. By employing RT-PCR,R-14 mRNA is shown to be expressed in human trabecular meshwork, but notin another ocular tissue, iris. R-14 reading frame was cloned intomammalian expression vectors and transiently as well as stably expressedin HEK293 cells. Using these cells, a protein of 40 kDa was identifiedto be encoded by the recombinant R-14 gene. Furthermore, using selectivepeptidic ligands, applicants have shown that inhibition of R-14 receptorresulted in reduction of basal cyclic AMP synthesis, but not basalphosphoinositide levels, in porcine trabecular meshwork and mostimportantly, reduction of basal intraocular pressure in rabbits, adultpig eyes and anesthetized piglets. Based on this information, R-14receptor is identified as a useful target for screening for ocularhypotensive drugs, which may be useful for treatment of conditionsassociated with elevated intraocular pressure such as glaucoma andrelated conditions.

Furthermore, compositions containing selective inhibitors of R-14receptor are described which could be potentially used as ocularhypotensive drugs and glaucoma therapeutics or as lead compounds towardsdevelopment of such drugs. Bioassays in which host cells (e.g. mammaliancells) containing recombinantly expressed R-14 are described which canbe used to screen chemical compound libraries to identify lead compoundsfor providing R-14 ligands which in turn can be optimized into ocularhypotensive drugs and glaucoma therapeutics.

Definitions

For convenience, the meaning of certain terms and phrases employed inthe specification, examples, and appended claims are provided below.

The term “agonist”, as used herein, is meant to refer to an agent thatpotentiates at least one aspect of R-14 bioactivity. R-14 bioactivitycan be increased for example, by stimulating the wild-type activity andsignal transduction, or enable the wild type R-14 protein to interactmore efficiently with other proteins which are involved in signaltransduction cascade.

“Antagonist” as used herein is meant to refer to an agent that inhibitsat least one R-14 bioactivity. An R-14 antagonist can be a compoundwhich inhibits or decreases the interaction between a R-14 protein andanother molecule, or decreases the synthesis and expression of R-14polypeptide or inhibits the bioactivity of R-14 molecule. The antagonistcan be a nucleic acid molecule, such as a dominant negative form ofR-14, an R-14 antisense molecule, a ribozyme capable of specificallyinteracting with R-14 mRNA, or molecules which bind to a R-14polypeptide e.g. peptides, antibodies, small molecules.

An “agent capable of modulating R-14 activity” refers to any compoundwhich when introduced into a system comprising an R-14 protein, iscapable of altering at least one aspect of R-14 activity or function.Such an agent may be a ligand of an R-14 protein, in further embodimentsan agonist or antagonist as defined above. Such an agent may actdirectly on an R-14 protein or indirectly by modulating a process oractivity which subsequently results in the modulation of R-14 activity.In certain systems (e.g. in vivo), such an agent may be a prodrug whichis metabolised to an active form at or prior to its arrival at the siteof action. Examples of R-14 activity are noted below.

The term “amino acid” as used herein includes both L and D isomers ofthe naturally occurring amino acids (Table 1) as well as othernonproteinaceous amino acids used in peptide chemistry to preparesynthetic analogs of peptides. Examples of naturally-occurring aminoacids are glycine, alanine, valine, leucine, isoleucine, serine,threonine, etc. whereas nonproteinaceous amino acids are norleucine,norvaline, cyclohexyl alanine, biphenyl alanine, homophenyl alanine,naphthyl alanine, pyridyl alanine, phenyl alanines substituted at theortho, para and meta positions with alkoxy, halogen or nitro groups etc.These compounds are known to persons versed in the art of peptidechemistry.

TABLE 1 Common notations of amino acids (L-amino acids by capitalletters and D-amino acids by small letters are represented byconvention; glycine does not have L/D forms) 3-Letter 1-Letter Full NameCode Code Aspartic Acid Asp D/d Glutamic Acid Glu E/e Lysine Lys K/kArginine Arg R/r Histidine His H/h Tyrosine Tyr Y/y Cysteine Cys C/cAsparagine Asn N/n Glutamine Gln Q/q Serine Ser S/s Threonine Thr T/tGlycine Gly G Alanine Ala A/a Valine Val V/v Leucine Leu L/l IsoleucineIle I/I Methionine Met M/m Proline Pro P/p Phenylalanine Phe F/fTryptophan Trp W/w

“Biological activity” or “bioactivity” or “activity” or “biologicalfunction”, which are used interchangeably, for the purposes herein meansa function that is directly or indirectly performed by an R-14polypeptide, or by any fragment thereof. In this instance, biologicalactivities of R-14 include binding to another molecule, interaction withother proteins, alterations in signal transduction such as guaninenucleotide binding by G_(α) proteins, calcium fluxes, cAMP synthesis,inositol phosphate synthesis, internalization of R-14 polypeptide,association with other intracellular proteins or coated pits in cellmembrane, alterations in intraocular pressure, elevation of aqueoushumor transit through the Schlemm's canal etc.

“Cells”, “host cells” or “recombinant host cells” are terms usedinterchangeably herein. It is understood that such terms refer not onlyto the particular cell but to all its progeny. Also within the scope ofthe term are cells of mammalian (e.g. human), amphibian, fungal (e.g.yeast), and bacterial (e.g. E. coli) origin.

A “fusion polypeptide” can be represented by the general formulaX-(R-14)-Y, wherein R-14 represents a portion of the polypeptide whichis derived from a R-14 polypeptide, and X and Y are amino acid sequencesand are independently present or absent in the fusion peptide.

As used herein, the term “nucleic acid” refers to polynucleotides oroligonucleotides such as deoxyribonucleic acid (DNA), and, whereappropriate, ribonucleic acid (RNA).

The terms “protein” and “polypeptide” are used interchangeably hereinwhen referring to a gene product.

The term “R-14 nucleic acid” refers to a nucleic acid capable ofencoding an R-14 protein, such as nucleic acids having SEQ ID NO. 1, aswell as fragments thereof, and sequences substantially identicalthereto.

The terms “R-14 polypeptide” and “R-14 protein” are intended toencompass polypeptides comprising the amino acid sequence shown as SEQID NO:2 or fragments, variants and homologs thereof.

“Homology” and “homologous” refers to sequence similarity between twopeptides or two nucleic acid molecules. Homology can be determined bycomparing each position in the aligned sequences. A degree of homologybetween nucleic acid or between amino acid sequences is a function ofthe number of identical or matching nucleotides or amino acids atpositions shared by the sequences. An “unrelated” or “non-homologous”sequence shares less than 40% identity, though preferably less thanabout 25% identity, with SEQ ID Nos:1 or 2 of the present invention.

Substantially complementary nucleic acids are nucleic acids in which the“complement” of one molecule is substantially identical to the othermolecule. Two nucleic acid or protein sequences are considered“substantially identical” if, when optimally aligned, they share atleast about 70% sequence identity. In alternative embodiments, sequenceidentity may for example be at least 75%, at least 80%, at least 85%, atleast 90%, or at least 95%. Optimal alignment of sequences forcomparisons of identity may be conducted using a variety of algorithms,such as the local homology algorithm of Smith and Waterman,1981, Adv.Appl. Math 2: 482, the homology alignment algorithm of Needleman andWunsch, 1970, J. Mol. Biol. 48:443, the search for similarity method ofPearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85: 2444, and thecomputerised implementations of these algorithms (such as GAP, BESTFIT,FASTA and TFASTA in the Wisconsin Genetics Software Package, GeneticsComputer Group, Madison, Wis., U.S.A.). Sequence identity may also bedetermined using the BLAST algorithm, described in Altschul et al.,1990, J. Mol. Biol. 215:403-10 (using the published default settings).Software for performing BLAST analysis may be available through theNational Center for Biotechnology Information (through the internet athttp://www.ncbi.nlm.nih.gov/). The BLAST algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence that either match or satisfysome positive-valued threshold score T when aligned with, a word of thesame length in a database sequence. T is referred to as theneighbourhood word score threshold. Initial neighbourhood word hits actas seeds for initiating searches to find longer HSPs. The word hits areextended in both directions along each sequence for as far as thecumulative alignment score can be increased. Extension of the word hitsin each direction is halted when the following parameters are met: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, Tand X determine the sensitivity and speed of the alignment. The BLASTprogram may use as defaults a word length (W) of 11, the BLOSUM62scoring matrix (Henikoff and Henikoff, 1992, Proc. Natl. Acad. Sci. USA89: 10915-10919) alignments (B) of 50, expectation (E) of 10 (or 1 or0.1 or 0.01 or 0.001 or 0.0001), M=5, N=4, and a comparison of bothstrands. One measure of the statistical similarity between two sequencesusing the BLAST algorithm is the smallest sum probability (P(N)), whichprovides an indication of the probability by which a match between twonucleotide or amino acid sequences would occur by chance. In alternativeembodiments of the invention, nucleotide or amino acid sequences areconsidered substantially identical if the smallest sum probability in acomparison of the test sequences is less than about 1, preferably lessthan about 0.1, more preferably less than about 0.01, and mostpreferably less than about 0.001.

An alternative indication that two nucleic acid sequences aresubstantially complementary is that the two sequences hybridize to eachother under moderately stringent, or preferably stringent, conditions.Hybridization to filter-bound sequences under moderately stringentconditions may, for example, be performed in 0.5 M NaHPO₄, 7% sodiumdodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.2×SSC/0.1%SDS at 42° C. (see Ausubel, et al. (eds), 1989, Current Protocols inMolecular Biology, Vol. 1, Green Publishing Associates, Inc., and JohnWiley & Sons, Inc., New York, at p. 2.10.3). Alternatively,hybridization to filter-bound sequences under stringent conditions may,for example, be performed in 0.5 M NaHPO₄, 7% SDS, 1 mM EDTA at 65° C.,and washing in 0.1×SSC/0.1% SDS at 68° C. (see Ausubel, et al. (eds),1989, supra). Hybridization conditions may be modified in accordancewith known methods depending on the sequence of interest (see Tijssen,1993, Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, New York). Generally, stringent conditionsare selected to be about 5° C. lower than the thermal melting point forthe specific sequence at a defined ionic strength and pH.

The term “interact” as used herein is meant to include detectablerelationships or associations (e.g. biochemical interactions) betweenmolecules, such as interaction between protein-protein, protein-nucleicacid, nucleic acid-nucleic acid, and protein-small molecule or nucleicacid-small molecule in nature.

The term “modulation” as used herein refers to both upregulation (i.e.,activation or stimulation (e.g., by agonizing or potentiating)) anddownregulation (i.e. inhibition or suppression (e.g., by antagonizing,decreasing or inhibiting)).

The term “peptide” is intended to mean a linear polymer containing atleast 2 amino acids to a maximum of 50 amino acids. In furtherembodiments the peptide contains 2 to 25 amino acids, 2 to 20 aminoacids, 2 to 15 amino acids, 2 to 10 amino acids or 8 amino acids. Theamino acids can be naturally-occuring or synthetically-derivedmolecules. Examples of such molecules are L-amino acids, D-amino acids,and synthetic analogues of natural amino acids including but not limitedto nonproteinaceous amino acids.

The term “peptidomimetic” refers to a molecule that mimics thestructural and/or functional features of a peptide. Persons skilled inthe art use variety of methods to derive peptidomimetics of a peptide:substitutions of individual amino acids with synthetic chemicalentities, nonproteinaceous amino acid analogues, deletions, additions ofamino acids, replacing one or more of amino acids in the peptide withscaffolds such as beta turn mimetics, or with known pharmacophores. Adescription of the general methods are given in Peptidomimetic protocols(Methods in molecular medicine Vol. 23) W. M. Kazmierski (ed.), HumanaPress and Advances in Amino Acid Mimetics and Peptidomimetics, Vols. 1 &2 A. Abell (Ed).

The term “recombinant protein” refers to a polypeptide of the presentinvention which is produced by recombinant DNA techniques, whereingenerally, DNA encoding a R-14 polypeptide is inserted into a suitableexpression vector which is in turn used to transform a host cell toproduce the heterologous protein. “Recombinant” means that something hasbeen recombined, so that when made in reference to a nucleic acidconstruct the term refers to a molecule that is comprised of nucleicacid sequences that are joined together or produced by means ofmolecular biological techniques. The term “recombinant” when made inreference to a protein or a polypeptide refers to a protein orpolypeptide molecule which is expressed using a recombinant nucleic acidconstruct created by means of molecular biological techniques. The term“recombinant” when made in reference to genetic composition refers to agamete or progeny or cell or genome with new combinations of allelesthat did not occur in the parental genomes. Recombinant nucleic acidconstructs may include a nucleotide sequence which is ligated to, or ismanipulated to become ligated to, a nucleic acid sequence to which it isnot ligated in nature, or to which it is ligated at a different locationin nature. Referring to a nucleic acid construct as ‘recombinant’therefore indicates that the nucleic acid molecule has been manipulatedusing genetic engineering, i.e. by human intervention. Recombinantnucleic acid constructs may for example be introduced into a host cellby transformation. Such recombinant nucleic acid constructs may includesequences derived from the same host cell species or from different hostcell species, which have been isolated and reintroduced into cells ofthe host species. Recombinant nucleic acid construct sequences maybecome integrated into a host cell genome, either as a result of theoriginal transformation of the host cells, or as the result ofsubsequent recombination and/or repair events.

The term “vector” refers to a nucleic acid molecule, which is capable oftransporting another nucleic acid to which it has been linked. One typeof preferred vector is an episome, i.e., a nucleic acid capable ofextra-chromosomal replication. Preferred vectors are those capable ofautonomous replication and/or expression of nucleic acids to which theyare linked. Vectors capable of directing the expression of genes towhich they are operatively linked are referred to herein as “expressionvectors”.

The recombinant expression vector of the present invention can beconstructed by standard techniques known to one of ordinary skill in theart and found, for example, in Sambrook et al. (1989) in MolecularCloning: A Laboratory Manual. A variety of strategies are available forligating fragments of DNA, the choice of which depends on the nature ofthe termini of the DNA fragments and can be readily determined bypersons skilled in the art. The vectors of the present invention mayalso contain other sequence elements to facilitate vector propagationand selection in bacteria and host cells. In addition, the vectors ofthe present invention may comprise a sequence of nucleotides for one ormore restriction endonuclease sites. Coding sequences such as forselectable markers and reporter genes are well known to persons skilledin the art.

A recombinant expression vector comprising a nucleic acid sequence ofthe present invention may be introduced into a host cell, which mayinclude a living cell capable of expressing the protein coding regionfrom the defined recombinant expression vector. The living cell mayinclude both a cultured cell and a cell within a living organism.Accordingly, the invention also provides host cells containing therecombinant expression vectors of the invention. The terms “host cell”and “recombinant host cell” are used interchangeably herein. Such termsrefer not only to the particular subject cell but to the progeny orpotential progeny of such a cell. Because certain modifications mayoccur in succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term as usedherein.

Vector DNA can be introduced into cells via conventional transformationor transfection techniques. The terms “transformation” and“transfection” refer to techniques for introducing foreign nucleic acidinto a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection,electroporation, microinjection and viral-mediated transfection.Suitable methods for transforming or transfecting host cells can forexample be found in Sambrook et al. (Molecular Cloning: A LaboratoryManual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), andother laboratory manuals. Methods for introducing DNA into mammaliancells in vivo are also known, and may be used to deliver the vector DNAof the invention to a subject for gene therapy for lowering intraocularpressure and/or for treating associated conditions, such as glaucoma.

“Transcriptional regulatory sequence/element” is a generic term thatrefers to DNA sequences, such as initiation and termination signals,enhancers, and promoters, splicing signals, polyadenylation signalswhich induce or control transcription of protein coding sequences withwhich they are operably linked. In the present invention, transcriptionof one of the R-14 genes is under the control of a promoter sequencewhich controls the expression of the recombinant gene in a cell-type. Afirst nucleic acid sequence is “operably-linked” with a second nucleicacid sequence when the first nucleic acid sequence is placed in afunctional relationship with the second nucleic acid sequence. Forinstance, a promoter is operably-linked to a coding sequence if thepromoter affects the transcription or expression of the codingsequences. Generally, operably-linked DNA sequences are contiguous and,where necessary to join two protein coding regions, in reading frame.However, since for example enhancers generally function when separatedfrom the promoters by several kilobases and intronic sequences may be ofvariable lengths, some polynucleotide elements may be operably-linkedbut not contiguous.

As used herein, the term “transfection” means the introduction of anucleic acid, e.g., via an expression vector, into a recipient cell bynucleic acid-mediated gene transfer.

A cell (e.g. a host cell or indicator cell), tissue, organ, or organisminto which has been introduced a foreign nucleic acid (e.g. exogenous orheterologous DNA [e.g. a DNA construct]), is considered “transformed”,“transfected”, or “transgenic”. A transgenic or transformed cell ororganism also includes progeny of the cell or organism and progenyproduced from a breeding program employing a transgenic organism as aparent and exhibiting an altered phenotype resulting from the presenceof a recombinant nucleic acid construct. A transgenic organism istherefore an organism that has been transformed with a heterologousnucleic acid, or the progeny of such an organism that includes thetransgene. The introduced DNA may be integrated into chromosomal DNA ofthe cell's genome, or alternatively may be maintained episomally (e.g.on a plasmid). Methods of transfection are well known in the art(Sambrook et al., 1989, supra; Ausubel et al., 1994 supra).

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (such as resistance to antibiotics) may be introducedinto the host cells along with the gene of interest. As used herein, theterm “selectable marker” is used broadly to refer to markers whichconfer an identifiable trait to the indicator cell. Non-limiting exampleof selectable markers include markers affecting viability, metabolism,proliferation, morphology and the like. Preferred selectable markersinclude those that confer resistance to drugs, such as G418, hygromycinand methotrexate. Nucleic acids encoding a selectable marker may beintroduced into a host cell on the same vector as that encoding thepeptide compound or may be introduced on a separate vector. Cells stablytransfected with the introduced nucleic acid may be identified by drugselection (cells that have incorporated the selectable marker gene willsurvive, while the other cells die).

A compound is “substantially pure” when it is separated from thecomponents that naturally accompany it. Typically, a compound issubstantially pure when it is at least 60%, more generally 75% or over90%, by weight, of the total material in a sample. Thus, for example, apolypeptide that is chemically synthesised or produced by recombinanttechnology will generally be substantially free from its naturallyassociated components. A nucleic acid molecule is substantially purewhen it is not immediately contiguous with (i.e., covalently linked to)the coding sequences with which it is normally contiguous in thenaturally occurring genome of the organism from which the DNA of theinvention is derived. A substantially pure compound can be obtained, forexample, by extraction from a natural source; by expression of arecombinant nucleic acid molecule encoding a polypeptide compound; or bychemical synthesis. Purity can be measured using any appropriate methodsuch as column chromatography, gel electrophoresis, HPLC, etc.

“Small molecule” as used herein, is meant to refer to a composition,which has a molecular weight of less than about 1 kD and most preferablyless than about 0.4 kD. Small molecules can be nucleotides, amino acids,peptides, peptidomimetics, carbohydrates, lipids or other organic(carbon containing) molecules. Many pharmaceutical companies haveextensive libraries of chemical and/or biological mixtures, oftenfungal, bacterial, or algal extracts, which can be screened with any ofthe assays of the invention to identify compounds that modulate an R-14bioactivity.

The term “alkyl” refers to the radical of saturated aliphatic groups,including straight chain alkyl groups, branched-chain alkyl groups,cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, andcycloalkyl substituted alkyl groups. Typical alkyl groups include, butare not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl,t-butyl, pentyl, isopentyl, hexyl, etc. The alkyl groups can be (C₁-C₆)alkyl, or, (C₁-C₃) alkyl. A “substituted alkyl” has substituentsreplacing a hydrogen on one or more carbons of the hydrocarbon backbone.Such substituents can include, for example, halogen, hydroxyl, carbonyl(such as carboxyl, ketones (including alkylcarbonyl and arylcarbonylgroups), and esters (including alkyloxycarbonyl and aryloxycarbonylgroups)), thiocarbonyl, acyloxy, alkoxyl, phosphoryl, phosphonate,phosphinate, amino, acylamino, amido, amidine, imino, cyano, nitro,azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl,sulfonamido, heterocyclyl, aralkyl, or an aromatic or heteroaromaticmoiety. The moieties substituted on the hydrocarbon chain can themselvesbe substituted, if appropriate. For instance, the substituents of asubstituted alkyl may include substituted and unsubstituted forms ofaminos, azidos, iminos, amidos, phosphoryls (including phosphonates andphosphinates), sulfonyls (including sulfates, sulfonamidos, sulfamoylsand sulfonates), and silyl groups, as well as ethers, alkylthios,carbonyls (including ketones, aldehydes, carboxylates, and esters),—CF₃, —CN and the like. Exemplary substituted alkyls are describedbelow. Cycloalkyls can be further substituted with alkyls, alkenyls,alkoxys, alkylthios, aminoalkyls, carbonyl-substituted alkyls, —CF₃,—CN, and the like.

The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond respectively.An “alkenyl” is an unsaturated branched, straight chain, or cyclichydrocarbon radical with at least one carbon-carbon double bond. Theradical can be in either the cis or trans conformation about the doublebond(s). Typical alkenyl groups include, but are not limited to,ethenyl, propenyl, isopropenyl, butenyl, isobutenyl, tert-butenyl,pentenyl, hexenyl, etc. An “alkynyl” is an unsaturated branched,straight chain, or cyclic hydrocarbon radical with at least onecarbon-carbon triple bond. Typical alkynyl groups include, but are notlimited to, ethynyl, propynyl, butynyl, isobutynyl, pentynyl, hexynyl,etc.

A “therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result, such as reduction of intraocular pressure andrelated disorders such as glaucoma. A therapeutically effective amountof a compound of the invention may vary according to factors such as thedisease state, age, sex, and weight of the individual, and the abilityof the compound to elicit a desired response in the individual. Dosageregimens may be adjusted to provide the optimum therapeutic response. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the compound are outweighed by thetherapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result, such aspreventing or reducing elevated intraocular pressure and in turnpreventing or treating related disorders such as glaucoma. Aprophylactically effective amount can be determined as described abovefor the therapeutically effective amount. For any particular subject,specific dosage regimens may be adjusted over time according to theindividual need and the professional judgement of the personadministering or supervising the administration of the compositions.

As used herein “pharmaceutically acceptable carrier” or “excipient”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike that are physiologically compatible. In one embodiment, the carrieris suitable for parenteral administration. Alternatively, the carriercan be suitable for intravenous, intraperitoneal, intramuscular,sublingual or oral administration. Pharmaceutically acceptable carriersinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersion. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active compound, use thereof inthe pharmaceutical compositions of the invention is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

R14 Nucleic Acids

As noted above, the present invention is based, in part, on thediscovery of a human gene, which encodes a human nucleic acid andencoding a protein referred to as “R-14”. In silico analysis showed thatR-14 could be an integral membrane protein containing seven prominenthydrophobic domains and many signature motifs of a G protein-coupledreceptor. The protein and nucleic acid of R-14 show strong homology tohuman, mouse and rat MAS-related G protein-coupled receptors by BLASTNand BLASTP analyses. A10.96 kb subclone of BAC RP11-206c1 (obtained fromSanger Center, UK) was completely sequenced and determined to containthe entire coding region of R-14 (SEQ ID NO:1). The human R-14 codingsequence is 0.966 kb in size and contains no introns. The 966 bp openreading frame (SEQ ID NO:1) encodes a 322 amino acid polypeptide (SEQ IDNO:2). BLASTN analysis of dBEST data base revealed near identity to theEST termed as HTMI-0025F1 (GenBank Acc.: BE439409) and the nucleic acidsequence is identical to HTMI-0025 (Gonzalez, P. et al., supra) with theexception of two nucleotide changes, A to C leading to Gln to Pro at661158 (NT_(—)009307.3 (Hs11_(—)9464).

The invention provides an isolated R-14 nucleic acid, homologs thereof,and portions thereof. Preferred nucleic acids have a sequence, which isat least about 60%, 65%, 70%, 75%, 80%, 85% and preferably 90% and 95%homologous with a nucleotide sequence of an R-14 gene, e.g., such as asequence shown in SEQ ID NO. 1. Preferred nucleic acids are vertebrateR-14 nucleic acids. Particularly preferred vertebrate R-14 nucleic acidsare mammalian, in an embodiment, human. In one embodiment, the preferrednucleic acid is a cDNA encoding a polypeptide having at least onebioactivity of the subject R-14 polypeptide.

R14 Polypeptides

The present invention features R-14 polypeptides which can be producedin and isolated from, cells or tissues in which the the polypeptide isnaturally expressed, or cells in which R-14 polypeptide is expressedusing gene transfer of recombinant R1-4 nucleic acids, either ascell-free extracts or purified membrane fractions. Functional forms ofthe subject polypeptides can be prepared as purified preparations byusing a cloned gene as described herein. Preferred R-14 proteins of theinvention have an amino acid sequence which is at least about 60%, 65%,70%, 75, 80%, 85%, 90%, or 95% identical or homologous to the amino acidsequence of SEQ ID NO:2. In a preferred embodiment, an R-14 protein ofthe present invention is a mammalian R-14 protein. In a particularlypreferred embodiment an R-14 protein is set forth as SEQ ID NO:2. Itwill be understood that certain post-translational modifications, e.g.,phosphorylation and the like, can increase the apparent molecular weightof the R-14 protein relative to the unmodified polypeptide chain.Protein isoforms encoded by splice variants of R-14 listed in SEQ IDNO:2 are also within the scope of the present invention. Such isoformsmay have additional biological activities from those possessed by theR-14 proteins specified by SEQ ID NO:2.

R14 Polypeptide Fusions, Mutant-Proteins and Homologs Thereof

In one aspect, the invention relates to R-14 polypeptides, either asenriched fractions of cells and tissues, or substantially purepreparations of naturally-occurring or recombinantly-produced orchemically-synthesized polypeptides. An R-14 polypeptide of theinvention can comprise a full length protein as set forth in SEQ ID NO:2or can comprises fusion proteins containing smaller fragmentscorresponding to one or more particular motifs/domains, or fragmentscomprising at least about 100, 125, 150, 175, 200, 225, 250, 275, 300amino acids in length. The subject R-14 protein also includes within itsscope modified proteins, e.g. proteins in which specific mutationsprevent post-translational modification, such as glycosylation,myristylation, palmitylation and phosphorylation of the protein, orwhich mutations prevent or enhance interaction of the R-14 mutantprotein with agonists, antagonists or intracellular proteins involved insignal transduction.

In further embodiments, R-14 polypeptides of the invention may comprisea fragment of the polypeptide of SEQ ID NO:2, as defined by a minimumnumber of consecutive amino acids thereof. Accordingly, in embodimentsthe invention relates to an R-14 polypeptide comprising at least 100,125, 150, 175, 200, 225, 250, 275, 278, 280, 290, 300, 305, 310, 315,320 consecutive amino acids of SEQ ID NO:2.

In another aspect, the, invention relates to a recombinant expressionsystem for producing an R-14 protein. For expression in cells, tissuesand animals, the nucleic acid as set in SEQ. ID NO. 1 was cloned into amammalian expression vector, in which R-14 nucleic acid was operablylinked to a transcriptional regulatory sequence, e.g., at least one of atranscriptional promoter (for constitutive expression), sequencesrequired for splicing and transcription termination. Such regulatorysequences in conjunction with a R-14 nucleic acid molecule provided auseful vector for gene expression. People skilled in the art could usesimilar strategies to express R-14 protein in prokaryotic and eukaryotichost cells transfected with appropriate expression vectors in vitro(e.g. cell culture) and in vivo (e.g. transgenic) methods for producingR-14 proteins.

The present invention further pertains to methods of producing thesubject R-14 polypeptides. For example, a “host cell” transfected with anucleic acid “vector” directing expression of a nucleotide sequenceencoding the subject polypeptides can be cultured under appropriateconditions to allow expression of the peptide to occur. Suitable mediafor cell culture are well known in the art. The recombinant R-14polypeptide can be isolated from cell culture medium, host cells, orboth using techniques known in the art for purifying proteins includingion-exchange chromatography, gel filtration chromatography,ultrafiltration, electrophoresis, and immunoaffinity purification withantibodies specific for such peptides. In a preferred embodiment, therecombinant R-14 polypeptide is a fusion protein containing a domainwhich facilitates its purification, such as polyhistidine fusion of R-14protein.

The invention also provides fusion proteins, e.g., R-14 conjugated togreen fluorescent protein or beta arrestin. Such fusion proteins canprovide detection of R-14 polypeptides in cells, tissues and organisms.Fusions of green fluorescent protein (GFP) to R-14 protein can be usedto locate and follow the dynamics of R-14, such as aggregation,association with other cellular proteins, internalization, trafficking,degradation in endocytotic vesicles, in living or fixed cells. R-14fusions of GFP and luciferase can be used to study and monitor dimer andoligomer formation, association with other signalling molecules.R-14-G_(α) protein fusions can be used to measure GTP binding andhydrolysis by the G protein in response to agonists or antagonists andthese methods, known to people skilled in the art, are used to screenand/or test small molecule compound libraries for agonist or antagonistactivity. These examples are presented to illustrate, but not to limitthe potential fusion partners and their uses in basic and appliedscientific studies.

Moreover, it will be generally appreciated that, under certaincircumstances, it may be advantageous to provide homologs of one of thesubject R-14 polypeptides, which function in a limited capacity as oneof either an R-14 agonist (mimetic) or an R-14 antagonist, in order topromote or inhibit only a subset of the biological activities of thenaturally-occurring form of the protein. Homologs of each of the subjectR-14 proteins can be generated by mutagenesis, such as by discrete pointmutation(s), or by truncation. For instance, mutation can give rise tohomologs which display elevated ligand-independent activity orsubstantially the same, or merely a subset of the biological activity ofthe R-14 polypeptide from which it was derived. Alternatively,antagonistic forms of the protein can be generated which are able toinhibit the function of the naturally occurring form of the protein,such as by competitively binding to an R-14 receptor.

R14 Activity and Assaying Said Activity

In one aspect, the invention provides methods for identifying a compoundthat can modulate R-14 “activity”. Such a method may entail determiningthe activity of an R-14 protein in the presence of a test or candidatecompound. Such a method may for, example be used to identify an R-14antagonist, which may be useful for lowering intraocular pressure, andfurther for treating a condition associated with elevated intraocularpressure, such as glaucoma and related conditions. Various aspects ofR-14 activity may be assayed in this regard, as noted herein and in theExamples.

In an embodiment, determining R-14 “activity” entails assaying aninteraction between an R-14 polypeptide and an R-14 binding partner, toidentify compounds that are capable of interfering with the interactionof R-14 and its binding partner, and thus the test compound may becapable of binding to an R-14 polypeptide. In an embodiment, such amethod includes the steps of (a) forming a mixture, which includes: (i)an R-14 polypeptide, (ii) an R-14 binding partner and (iii) a testcompound; and (b) detecting interaction of the R-14 polypeptide and theR-14 binding partner or alteration in at least one aspect of R-14polypeptide “activity”. A statistically significant change (potentiationor inhibition) in R-14 activity in the presence of the test compound,relative to that in the absence of the test compound, indicates apotential agonist or antagonist (inhibitor) respectively of R-14bioactivity for the test compound. The reaction mixture can be acell-free protein preparation, e.g., a reconstituted protein mixture ora cell lysate or purified cell constituents, or a cultured cellrecombinantly expressing the R-14 polypeptide or fragments thereof.People skilled in the art can use such a competitive binding assay todetect the interaction between an R-14 polypeptide and a, R-14 bindingpartner. In an embodiment, at least one of the R-14 polypeptide and theR-14 binding partner comprises a detectable label, and interaction ofthe R-14 and R-14 binding partner is quantified by detecting the labelin the complex. The detectable label can be, e.g., a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor.

Cell-Free Assays

Cell-free assays can be used to identify compounds which are capable ofinteracting with an R-14 protein, thereby modify the activity of theR-14 protein. Such a compound can, e.g., modify the structure of, anR-14 protein and thereby affect its activity. Cell-free assays can alsobe used to identify compounds which modulate the interaction between anR-14 protein and a R-14 binding partner. In a preferred embodiment,cell-free assays for identifying such compounds consist essentially in areaction mixture containing an R-14 protein, R-14 binding partner and atest compound. A test compound can be, e.g., a derivative of a R-14polypeptide or R-14 binding partner.

Accordingly, one exemplary screening assay of the present inventionincludes the steps of (a) forming a reaction mixture including: (i) anR-14 polypeptide, (ii) an R-14 binding partner, and (iii) a testcompound; and (b) detecting interaction of the R-14 and the R-14 bindingprotein. For detection purposes, the binding partner can be labelledwith a specific marker such as a radionuclide, or a fluorescent compoundor an enzyme. Interaction of a test compound with an R-14 protein orfragment thereof can then be detected by determining the level of themarker label after an incubation step and a washing step. The R-14polypeptide and R-14 binding partner can be produced recombinantly,purified from a source, e.g., plasma, or chemically synthesized, asdescribed herein. A statistically significant change (potentiation orinhibition) in the interaction of the R-14 and R-14 binding protein inthe presence of the test compound, relative to the interaction in theabsence of the test compound, indicates a potential agonist (mimetic orpotentiator) or antagonist (inhibitor) of R-14 bioactivity for the testcompound. Radiolabelled samples are counted and quantified byscintillation spectrophotometry. Binding ligands can be conjugated toenzymes such as acetylcholine esterase and bound R-14-binding partnercan be quantified by enzyme assay.

Cell-free assays can also be used to identify compounds which interactwith an R-14 protein and modulate an activity of an R-14 protein.Accordingly, in one embodiment, an R-14 protein is contacted with a testcompound and the bioactivity of R-14 is monitored. The bioactivity ofR-14 protein in cell-free assays include, GTP binding, GTP hydrolysis,Dissociation of G_(□) proteins, adenylate cyclase activation,phospholipase (A2, beta, gamma and D isoforms) activation, phospholipidhydrolysis, cAMP synthesis etc. and the methods of measuring thesechanges in the bioactivity of R-14 protein are well known to thoseskilled in the art.

Cell Based Assays

In addition to cell-free assays, such as described above, R-14 proteinsas provided by the present invention, facilitate the generation ofcell-based assays, e.g., for identifying an agent capable of modulatingR-14 activity, such as small molecule agonists or antagonists. Cellbased assays can be used, for example, to identify compounds whichmodulate the bioactivity of R-14 protein, expression of an R-14 gene orthose genes that are induced or suppressed in response to increased ordecreased bioactivity of R-14 protein. Accordingly, in one embodiment, acell which is capable of producing R-14 is incubated with a testcompound in the presence or absence of a natural or syntheticagonist/antagonist of R-14 and the bioactivity of R-14 is measured. Theresultant alterations in the bioactivity of R-14 are compared to controlR-14 producing cells which have not been contacted with the testcompound. These measurements are used to assess the potency, affinity,action of the test compound towards modulating R-14 bioactivity.

A particular embodiment of the present invention is that cell-free andcell-based assays involving the use of R-14 protein as set forth in SEQID NO. 2, could become an integral part of a screening system toevaluate and select small molecules which can be optimized to be used astherapeutics for lowering intraocular pressure and in the treatment ofglaucoma.

Kits

The invention further provides kits for use in diagnostics or screeningmethods for providing R-14 binding partners or glaucoma therapeutics.For example, the kit can comprise (1) a labeled R-14 binding partnerand/or (2) cell-free lysates or cellular fractions including membranesisolated from R-14 expressing host cells or R-14-expressing tissues orwhole cells expressing R-14 protein naturally or by recombinant DNAmethods and/or (3) an agent capable of detecting R-14 protein or mRNAand/or (4) means for determining the amount of R-14 protein, mRNA orbioactivity and the means for comparing the amount of R-14 protein, mRNAor bioactivity in the sample with a standard. The compound or agent canbe packaged in a suitable container. The kit can further compriseinstructions for using the kit to detect R-14 mRNA or protein or itsbioactivity. Such a kit can comprise, e.g., one or more nucleic acidprobes capable of hybridizing specifically to at least a portion of anR-14 gene or allelic variant thereof, or mutated form thereof.

R14 as a Drug Target in Intraocular Hypertension

Applicants' results described herein indicate that R-14 proteinrepresents a drug target, i.e. with a view to provide R-14 modulatorswhich may be useful for lowering intraocular pressure and thus may beuseful for the treatment of conditions associated with elevatedintraocular pressure, such as glaucoma and related conditions. In a morepreferred embodiment, the invention provides a method by which R-14 geneand protein can be expressed in host cells such as mammalian cells, andthe cells be used for small molecule or peptide lead compound discoveryin order to provide pharmaceutical compositions useful in treatingpatients diagnosed with elevated intraocular pressure and relatedconditions such as glaucoma.

R14 Antagonists, Pharmaceutical Compositions, Uses Thereof

Applicants have further identified and characterized agents (e.g.peptide compounds) capable of modulating R-14 activity, e.g. can act aspeptide antagonists of R-14 activity. Accordingly, the inventionprovides peptide compounds for use in lowering intraocular pressure. Ina further embodiment, the invention provides peptide compounds for usein the treatment of a condition associated with elevated intraocularpressure such as glaucoma and related conditions. The invention furtherprovides a method of lowering intraocular pressure in a subject and amethod for the treatment of a condition associated with elevatedintraocular pressure such as glaucoma and related conditions, themethods comprising administering an effective amount of apeptide/peptide compound of the invention, or a composition comprising apeptide of the invention, to the subject, e.g. a subject in needthereof. In an embodiment, the subject is a mammal, in a furtherembodiment, a human.

Therefore, in an aspect, the invention provides a substantially purepeptide compound of Formula I:Z₁-X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-Z₂  Iwherein:

-   X₁ is selected from the group consisting of Phe, His, Ile and    related amino acids;-   X₂ is selected from the group consisting of Ser, Ile, Phe and    related amino acids-   X₃ is selected from the group consisting of Leu, Ile, Asp and    related amino acids-   X₄ is selected from the group consisting of Thr, Cys, Ser and    related amino acids possessing side chains containing sulfhydryl,    hydroxyl or H-bond forming groups;-   X₅ is selected from the group consisting of Gln, Ser, Thr and    related amino acids;-   X₆ is selected from the group consisting of Lys, Pro, Glu and    related amino acids;-   X₇ is selected from the group consisting of Tyr, Leu, Cys and    related amino acids;-   X₈ is selected from the group consisting of Cys, Arg, Trp and    related amino acids;-   Z₁ is an N-terminal group of the formula H₂N—, RHN— or, RRN—;-   Z₂ is a C-terminal group of the formula —C(O)OH, —C(O)R, —C(O)OR,    —C(O)NHR, —C(O)NRR;-   R at each occurrence is independently selected from (C₁-C₆) alkyl,    (C₁-C₆) alkenyl, (C₁-C₆) alkynyl, substituted (C₁-C₆) alkyl,    substituted (C₁-C₆) alkenyl, or substituted (C₁-C₆) alkynyl; and-   “—” is a covalent linkage.

In a further embodiment, Z₁ is an N-terminal group selected from thegroup consisting of a proton, a sequence of 1-3 amino acids, or ablocking group such as a carbamate group, an acyl group composed of ahydrophobic moiety such as cyclohexyl, phenyl, benzyl, short chainlinear and branched alkyl groups of 1-8 carbons. In a furtherembodiment, Z₂ is a carboxy-terminal group selected from the groupconsisting of a proton, NH₂, 1-3 amino acids as well as arylalkyl aminessuch as benzylamine, phenylethylamine, phenylpropylamine, and aliphaticamines possessing short chain linear and branched alkyl groups of 1 to 8carbons.

The invention further provides a substantially pure synthetic peptidecompound or recombinant peptide compound, said compound having a domainof Formula II:-X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-  IIwherein X₁, X₂, X₃, X₄, X₅, X₆, X₇, X₈ and “—” are defined as above.

In embodiments, the peptide compound comprises a sequence selected fromthe group consisting of:

-   NH₂-Phe Ser Leu Thr Gln Lys Tyr Cys-OH (FSLTQKYC; SEQ ID NO:3);-   NH₂-His Ile Ile Cys Ser Pro Leu Arg-OH (HIICSPLR; SEQ ID NO:4); and-   NH₂-Ile Phe Asp Ser Thr Gln-Cys Trp-OH (IFDSTECW; SEQ ID NO:5).

The invention further provides an active fragment of the above notedpeptides. “Active fragment” refers to a fragment of a peptide of theinvention which is capable of modulating the activity of an R-14protein, wherein said R-14 protein comprises a polypeptide selected fromthe group consisting of:

-   (a) the polypeptide of SEQ ID NO. 2; and-   (b) a polypeptide encoded by a first nucleic acid that is    substantially identical to a second nucleic acid capable of encoding    the polypeptide of SEQ ID NO. 2.

The invention further provides derivatives of the above (SEQ ID NOs:3, 4and 5) which may be synthetic polypeptides containing conservativesubstitutions of individual amino acids, and peptidomimetics thereof.

The invention further provides a recombinant expression system, vectorsand host cells, such as those described above, for theexpression/production of a peptide comprising a peptide of the invention(such as those set forth in SEQ ID NOs. 3, 4 and 5), using for exampleculture media, production, isolation and purification methods such asthose described above. Such vectors comprise a nucleic acid sequencecapable of encoding such a peptide operably linked to a transcriptionalregulatory sequence. In an embodiment, the peptide is a fusion peptidecontaining a domain which facilitates its purification, such as apolyhistidine domain.

The invention also aims to provide a pharmaceutical compositioncomprising a peptidic or peptidomimetic compound with a pharmaceuticallyacceptable carrier, wherein said compound is capable of modulating, inan embodiment inhibiting at least one aspect of R-14 polypeptidebioactivity.

The invention also aims to provide a pharmaceutical compositioncontaining a peptidic or peptidomimetic compound with a pharmaceuticallyacceptable carrier, wherein said compound is capable of decreasingintraocular pressure.

Also within the scope of the invention are pharmaceutical compositionsfor treating patients diagnosed with increased intraocular pressureand/or glaucoma, comprising administering (e.g., either locally orsystemically) to a subject, a pharmaceutically effective amount of acomposition comprising a compound capable of modulating at least oneaspect of R-14 bioactivity.

R14 Antagonists, Active Fragments, Peptidomimetics Thereof

The invention also provides for reduction of the fragments of R-14antagonists to generate mimetics, e.g., peptide or non-peptide agents,such as small molecules, which are agonistic or antagonistic of R-14protein activity.

In order to improve the R-14 antagonists described in this invention fortherapeutic use, several modifications of the peptide can be made bysubstituting a first amino acid with a “related amino acid” which is asecond amino acid related to the first amino acid by either structure orfunction of the side chain: aromatic, aliphatic, positively- ornegatively-charged. Examples of related amino acids are provided inTables 2 and 3 below.

TABLE 2 Examples of related amino acids Residue Substitution Ala Gly;Ser Arg Lys Asn Gln; His Asp Glu Cys Ser Gln Asn Glu Asp Gly Ala; ProHis Asn; Gln Ile Leu; Val Leu Ile; Val Lys Arg Met Leu; Ile; Val PheMet; Leu; Tyr Ser Thr Thr Ser Trp Tyr; His; Phe Tyr Trp; Phe Val Ile;Leu Pro Ala; Gly

Alternatively, another group of substitutions of the R14 antagonists ofthe present invention are those in which at least one amino acid residuehas been removed and a different residue inserted in its place accordingto the following Table 2. Another group of substitutions are definedherein as exchanges within one of the following five groups:

TABLE 3 Relations among amino acids Small aliphatic, nonpolar or Ala,Ser, Thr, slightly polar residues (Pro, Gly) Polar, negatively chargedAsp, Asn, Glu, residues and their amides Gln Polar, positively chargedHis, Arg, Lys residues Large aliphatic, nonpolar Met, Leu, Ile, residuesVal, (Cys) Aromatic residues Phe, Tyr, Trp

The three amino acid residues in parentheses above have special roles inprotein architecture. Gly is the only residue lacking any side chain andthus imparts flexibility to the chain. This however tends to promote theformation of secondary structure other than alpha-helical. Pro, becauseof its unusual geometry, tightly constrains the chain. It generallytends to promote beta turn-like structures. Cys is capable ofparticipating in disulfide bond formation. Tyr, because of its hydrogenbonding potential, has significant kinship with Ser, and Thr, etc.

In addition, any amino acid representing a component of the saidpeptides can be replaced by the same amino acid but of the oppositechirality. Thus, any amino acid naturally occurring in theL-configuration (which may also be referred to as the R or S, dependingupon the structure of the chemical entity) may be replaced with an aminoacid of the same chemical structural type, but of the oppositechirality, generally referred to as the D-amino acid but which canadditionally be referred to as the R- or the S-, depending upon itscomposition and chemical configuration. Additional variations include b-and g-amino acids that provide different spatial arrangement of chemicalgroups.

In addition to the substitutions outlined above, synthetic amino acidsthat provide similar side chain functionality can be introduced in tothe peptide. For example, aromatic amino acids may be replaced with D-or L-naphthylalanine, D- or L-Phenylglycine, D- or L-2-thienylalanine,D- or L-1-, 2-, 3- or 4-pyrenylalanine, D- or L-3-thienylalanine, D- orL-(2-pyridinyl)-alanine, D- or L-(3-pyridinyl)-alanine, D- orL-(2-pyrazinyl)-alanine, D- or L-(4-isopropyl)-phenylglycine,D-(trifluoromethyl)-phenylglycine, D-(trifluoromethyl)-phenylalanine,D-p-fluorophenylalanine, D- or L-p-biphenylalanine D- orL-p-methoxybiphenylalanine, D- or L-2-indole(alkyl)alanines, and D- orL-alkylalanines where alkyl may be substituted or unsubstituted methyl,ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl, iso-pentylgroups. Non-carboxylate amino acids can be made to possess negativecharge, such as the non-limiting examples of phosphono- or sulfated(e.g. —SO₃H) amino acids.

Other substitutions may include unnatural alkylated amino acids whichare made by combining an alkyl group with any natural amino acid. Basicnatural amino acids such as lysine, arginine may be substituted withalkyl groups at NH₂. Others are nitrile derivatives (e.g., containingthe CN-moiety in place of CONH₂) of asparagine or glutamine, andsulfoxide derivative of methionine. In addition, any amide linkage inthe peptide can be replaced by a ketomethylene, hydroxyethyl,ethyl/reduced amide, thioamide or reversed amide moieties, e.g.(—C═O)—CH₂—), (—CHOH)—CH₂—), (CH₂—CH₂—), (—C═S)—NH—), or (—NH—(—C═O) for(—C═O)—NH—).

Compounds of the invention can be prepared, for example, by replacing,deleting, or inserting an amino acid residue of a peptide compound ordomain of the invention, with other conservative amino acid residues,i.e., residues having similar physical, biological, or chemicalproperties, and screening for biological function. It is well known inthe art that some modifications and changes can be made in the structureof a polypeptide without substantially altering the biological functionof that peptide, to obtain a biologically equivalent polypeptide. Thepeptides, ligands and domains of the present invention also extend tobiologically equivalent peptides, ligands and domains that differ from aportion of the sequence of novel ligands of the present invention byconservative amino acid substitutions. As used herein, the term“conserved amino acid substitutions” refers to the substitution of oneamino acid for another at a given location in the peptide, where thesubstitution can be made without substantial loss of the relevantfunction. In making such changes, substitutions of like amino acidresidues can be made on the basis of relative similarity of side-chainsubstituents, for example, their size, charge, hydrophobicity,hydrophilicity, and the like, and such substitutions may be assayed fortheir effect on the function of the peptide by routine testing.

In some embodiments, conserved amino acid substitutions may be madewhere an amino acid residue is substituted for another having a similarhydrophilicity value (e.g., within a value of plus or minus 2.0), wherethe following may be an amino acid having a hydropathic index of about−1.6 such as Tyr (−1.3) or Pro (−1.6)s are assigned to amino acidresidues (as detailed in U.S. Pat. No. 4,554,101, incorporated herein byreference): Arg (+3.0); Lys (+3.0); Asp (+3.0); Glu (+3.0); Ser (+0.3);Asn (+0.2); Gln (+0.2); Gly (0); Pro (−0.5); Thr (−0.4); Ala (−0.5); His(−0.5); Cys (−1.0); Met (−1.3); Val (−1.5); Leu (−1.8); Ile (−1.8); Tyr(−2.3); Phe (−2.5); and Trp (−3.4).

In alternative embodiments, conserved amino acid substitutions may bemade where an amino acid residue is substituted for another having asimilar hydropathic index (e.g., within a value of plus or minus 2.0).In such embodiments, each amino acid residue may be assigned ahydropathic index on the basis of its hydrophobicity and chargecharacteristics, as follows: Ile (+4.5); Val (+4.2); Leu (+3.8); Phe(+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); Gly (−0.4); Thr (−0.7); Ser(−0.8); Trp (−0.9); Tyr (−1.3); Pro (−1.6); His (−3.2); Glu (−3.5); Gln(−3.5); Asp (−3.5); Asn (−3.5); Lys (−3.9); and Arg (−4.5).

In alternative embodiments, conserved amino acid substitutions may bemade where an amino acid residue is substituted for another in the sameclass, where the amino acids are divided into non-polar, acidic, basicand neutral classes, as follows: non-polar: Ala, Val, Leu, Ile, Phe,Trp, Pro, Met; acidic: Asp, Glu; basic: Lys, Arg, His; neutral: Gly,Ser, Thr, Cys, Asn, Gln, Tyr.

Conservative amino acid changes can include the substitution of anL-amino acid by the corresponding D-amino acid, by a conservativeD-amino acid, or by a naturally-occurring, non-genetically encoded formof amino acid, as well as a conservative substitution of an L-aminoacid. Naturally-occurring non-genetically encoded amino acids includebeta-alanine, 3-amino-propionic acid, 2,3-diamino propionic acid,alpha-aminoisobutyric acid, 4-amino-butyric acid, N-methylglycine(sarcosine), hydroxyproline, ornithine, citrulline, t-butylalanine,t-butylglycine, N-methylisoleucine, phenylglycine, cyclohexylalanine,norleucine, norvaline, 2-napthylalanine, pyridylalanine, 3-benzothienylalanine, 4-chlorophenylalanine, 2-fluorophenylalanine,3-fluorophenylalanine, 4-fluorophenylalanine, penicillamine,1,2,3,4-tetrahydro-isoquinoline-3-carboxylix acid,beta-2-thienylalanine, methionine sulfoxide, homoarginine, N-acetyllysine, 2-amino butyric acid, 2-amino butyric acid, 2,4,-diamino butyricacid, p-aminophenylalanine, N-methylvaline, homocysteine, homoserine,cysteic acid, epsilon-amino hexanoic acid, delta-amino valeric acid, or2,3-diaminobutyric acid.

In alternative embodiments, conservative amino acid changes includechanges based on considerations of hydrophilicity or hydrophobicity,size or volume, or charge. Amino acids can be generally characterized ashydrophobic or hydrophilic, depending primarily on the properties of theamino acid side chain. A hydrophobic amino acid exhibits ahydrophobicity of greater than zero, and a hydrophilic amino acidexhibits a hydrophilicity of less than zero, based on the normalizedconsensus hydrophobicity scale of Eisenberg et al. (J. Mol. Bio.179:125-142, 1984). Genetically encoded hydrophobic amino acids includeGly, Ala, Phe, Val, Leu, Ile, Pro, Met and Trp, and genetically, encodedhydrophilic amino acids include Thr, His, Glu, Gln, Asp, Arg, Ser, andLys. Non-genetically encoded hydrophobic amino acids includet-butylalanine, while non-genetically encoded hydrophilic amino acidsinclude citrulline and homocysteine.

Hydrophobic or hydrophilic amino acids can be further subdivided basedon the characteristics of their side chains. For example, an aromaticamino acid is a hydrophobic amino acid with a side chain containing atleast one aromatic or heteroaromatic ring, which may contain one or moresubstituents such as —OH, —SH, —CN, —F, —Cl, —Br, —I, —NO₂, —NO, —NH₂,—NHR, —NRR, —C(O)R, —C(O)OH, —C(O)OR, —C(O)NH₂, —C(O)NHR, —C(O)NRR;etc., where R is independently (C₁-C₆) alkyl, substituted (C₁-C₆) alkyl,(C₁-C₆) alkenyl, substituted (C₁-C₆) alkenyl, (C₁-C₆) alkynyl,substituted (C₁-C₆) alkynyl, (C₅-C₂₀) aryl, substituted (C₅-C₂₀) aryl,(C₆-C₂₆) alkaryl, substituted (C₆-C₂₆) alkaryl, 5-20 memberedheteroaryl, substituted 5-20 membered heteroaryl, 6-26 memberedalkheteroaryl or substituted 6-26 membered alkheteroaryl. Geneticallyencoded aromatic amino acids include Phe, Tyr, and Tryp, whilenon-genetically encoded aromatic amino acids include phenylglycine,2-napthylalanine, beta-2-thienylalanine,1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid,4-chlorophenylalanine, 2-fluorophenylalanine3-fluorophenylalanine, and4-fluorophenylalanine.

An apolar amino acid is a hydrophobic amino acid with a side chain thatis uncharged at physiological pH and which has bonds in which a pair ofelectrons shared in common by two atoms is generally held, equally byeach of the two atoms (i.e., the side chain is not polar). Geneticallyencoded apolar amino acids include Gly, Leu, Val, Ile, Ala, and Met,while non-genetically encoded apolar amino acids includecyclohexylalanine. Apolar amino acids can be further subdivided toinclude aliphatic amino acids, which is a hydrophobic amino acid havingan aliphatic hydrocarbon side chain. Genetically encoded aliphatic aminoacids include Ala, Leu, Val, and Ile, while non-genetically encodedaliphatic amino acids include norleucine.

A polar amino acid is a hydrophilic amino acid with a side chain that isuncharged at physiological pH, but which has one bond in which the pairof electrons shared in common by two atoms is held more closely by oneof the atoms. Genetically encoded polar amino acids include Ser, Thr,Asn, and Gln, while non-genetically encoded polar amino acids includecitrulline, N-acetyl lysine, and methionine sulfoxide.

An acidic amino acid is a hydrophilic amino acid with a side chain pKavalue of less than 7. Acidic amino acids typically have negativelycharged side chains at physiological pH due to loss of a hydrogen ion.Genetically encoded acidic amino acids include Asp and Glu. A basicamino acid is a hydrophilic amino acid with a side chain pKa value ofgreater than 7. Basic amino acids typically have positively charged sidechains at physiological pH due to association with hydronium ion.Genetically encoded basic amino acids include Arg, Lys, and His, whilenon-genetically encoded basic amino acids include the non-cyclic aminoacids ornithine, 2,3,-diaminopropionic acid, 2,4-diaminobutyric acid,and homoarginine.

The above classifications are not absolute and an amino acid may beclassified in more than one category. In addition, amino acids can beclassified based on known behaviour and or characteristic chemical,physical, or biological properties based on specified assays or ascompared with previously identified amino acids. Amino acids can alsoinclude bifunctional moieties having amino acid-like side chains.

Conservative changes can also include the substitution of a chemicallyderivatised moiety for a non-derivatised residue, by for example,reaction of a functional side group of an amino acid. Thus, thesesubstitutions can include compounds whose free amino groups have beenderivatised to amine hydrochlorides, p-toluene sulfonyl groups,carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups orformyl groups. Similarly, free carboxyl groups can be derivatized toform salts, methyl and ethyl esters or other types of esters orhydrazides, and side chains can be derivatized to form O-acyl or O-alkylderivatives for, free hydroxyl groups or N-im-benzylhistidine for theimidazole nitrogen of histidine. Peptide analogues also include aminoacids that have been chemically altered, for example, by methylation, byamidation of the C-terminal amino acid by an alkylamine such asethylamine, ethanolamine, or ethylene diamine, or acylation ormethylation of an amino acid side chain (such as acylation of theepsilon amino group of lysine). Peptide analogues can also includereplacement of the amide linkage in the peptide with a substituted amide(for example, groups of the formula —C(O)—NR, where R is (C₁-C₆) alkyl,(C₁-C₆) alkenyl, (C₁-C₆) alkynyl, substituted (C₁-C₆) alkyl, substituted(C₁-C₆) alkenyl, or substituted (C₁-C₆) alkynyl) or isostere of an amidelinkage (for example, —CH₂NH—, —CH₂S, —CH₂CH₂—, —CH═CH— (cis and trans),—C(O)CH₂—, —CH(OH)CH₂—, or —CH₂SO—).

In order to improve the pharmaceutical characteristics of the R-14antagonists, the size of the peptides can be reduced by deleting one ormore amino acids and use amino acid mimetics or dipeptide mimicscontaining non-peptide bonds. Examples of using molecular scaffolds suchas benzodiazepine, azepine, substituted gamma lactam rings,keto-methylene pseudopeptides, β-turn dipeptide cores andβ-aminoalcohols for these purposes are known to peptide chemists and aredescribed in in Peptidomimetic protocols (Methods in molecular medicineVol. 23) W. M. Kazmierski (ed.), Humana Press and Advances in Amino AcidMimetics and Peptidomimetics, Vols. 1 & 2 A. Abell (Ed).

Covalent modifications of the peptide are thus included within the scopeof the present invention. Such modifications may be introduced into theR-14 antagonists by reacting targeted amino acid residues of thepolypeptide with an organic derivatizing agent that is capable ofreacting with selected side chains or terminal residues. The followingexamples of chemical derivatives are provided by way of illustration andnot by way of limitation. Cysteinyl residues may be reacted withalpha-haloacetates (and corresponding amines), such as 2-chloroaceticacid or chloroacetamide, to give carboxymethyl or carboxyamidomethylderivatives. Histidyl residues may be derivatized by reaction withcompounds such as diethylprocarbonate e.g., at pH 5.5-7.0 because thisagent is relatively specific for the histidyl side chain, andpara-bromophenacyl bromide may also be used; e.g., where the reaction ispreferably performed in 0.1M sodium cacodylate at pH 6.0. Lysinyl andamino terminal residues may be reacted with compounds such as succinicor other carboxylic acid anhydrides. Other suitable reagents forderivatizing alpha-amino-containing residues include compounds such asimidoesters/e.g. as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues may be modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin according to known method steps.Derivatization of arginine residues requires that the reaction beperformed in alkaline conditions because of the high pKa of theguanidine functional group. Furthermore, these reagents may react withthe groups of lysine as well as the arginine epsilon-amino group. Thespecific modification of tyrosinyl residues per se is well-known, suchas for introducing spectral labels into tyrosinyl residues by reactionwith aromatic diazonium compounds or tetranitromethane. N-acetylimidazoland tetranitromethane may be used to form O-acetyl tyrosinyl species and3-nitro derivatives, respectively.

Carboxyl side groups (aspartyl or glutamyl) may be selectively modifiedby reaction with carbodiimides (R′—N═C═N—R′) such as1-cyclohexyl-3-(2-morpholinyl-(4-ethyl)carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermoreaspartyl and glutamyl residues may be converted to asparaginyl andglutaminyl residues by reaction with ammonium ions. Glutaminyl andasparaginyl residues may be frequently deamidated to the correspondingglutamyl and aspartyl residues. Other modifications of the peptides inthe present invention may include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the alpha-amino groups of lysine, arginine, and histidineside chains acetylation of the N-terminal amine, methylation of mainchain amide residues (or substitution with N-methyl amino acids) and, insome instances, amidation of the C-terminal carboxyl groups, accordingto known method steps.

Covalent attachment of fatty acids (C6-C18) to the peptides conferadditional biological properties such as protease resistance, plasmaprotein binding, increased plasma half life, intracellular penetrationetc. The above description of modification of a R14 antagonist peptidesdoes not limit the scope of the approaches nor the possiblemodifications that can be engineered.

Peptides or peptide analogues can be synthesised by standard chemicaltechniques, for example, by automated synthesis using solution or solidphase synthesis methodology. Automated peptide synthesisers arecommercially available and use techniques well known in the art.Peptides and peptide analogues can also be prepared using recombinantDNA technology using standard methods. Accordingly, the inventionfurther provides nucleic acids that encode peptide compounds of theinvention. Such nucleic acids may be introduced into cells forexpression using standard recombinant techniques for stable or transientexpression. Nucleic acid molecules of the invention may include anychain of two or more nucleotides including naturally occurring ornon-naturally occurring nucleotides or nucleotide analogues.

Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject having elevated intraocular pressureand/or glaucoma and related conditions. Therefore, the invention furtherprovides a method for lowering intraocular pressure or for treating acondition associated with elevated intraocular pressure, such asglaucoma and related conditions, in a subject, the method comprisingadministration of an agent which is capable of R-14 antagonist activity.Administration of a prophylactic agent can occur prior to themanifestation of symptoms characteristic of the R-14 aberrancy, suchthat glaucoma is prevented or, alternatively, its progression delayed.In general, the prophylactic or therapeutic methods compriseadministering to the subject an effective amount of a compound which iscapable of antagonizing a wildtype R-14 activity or agonizing a mutant(defective) R-14 activity. Examples of suitable compounds include theantagonists, agonists or homologues described in detail herein.

Effective Dose

Toxicity and therapeutic efficacy of agents capable of modulating R-14activity, such as R-14 agonists or antagonists, can be determined bystandard pharmaceutical procedures in experimental animals, e.g., fordetermining The LD₅₀ (The Dose Lethal To 50% Of The Population) and TheED₅₀ (the dose therapeutically effective in 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex and it can be expressed as the ratio LD₅₀/ED₅₀. Compounds whichexhibit large therapeutic induces are preferred. The dosage of suchcompounds lies preferably within a range of circulating concentrationsthat include the ED₅₀ with little or no toxicity. The dosage may varywithin this range depending upon the dosage form employed and the routeof administration utilized. A dose may be formulated in animal models toachieve a circulating plasma concentration range that includes the IC₅₀(i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) determined in in vitro and ex vivoassays and animal studies. Such information can be used to moreaccurately determine useful doses in humans. Levels of R-14 therapeuticsin plasma may be measured, for example, by high performance liquidchromatography. The effective dose of a R-14 therapeutic (agonist orantagonist) could be 0.01 micrograms-100 mg and is determined by theroute of administration, pharmaceutical preparation and the mode ofdelivery.

Formulation and Use

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers or excipients. Thus, the compoundsand their physiologically acceptable salts and solvates may beformulated for administration by, for example, injection, inhalation(either through the mouth or the nose) or oral, buccal, parenteral orrectal administration. Techniques and formulations generally may befound in Reminington's Pharmaceutical Sciences, Meade Publishing Co.,Easton, Pa.). For topical administration, R-14 therapeutics of theinvention are formulated into solutions, ointments, salves, gels, orcreams as generally known in the art. For example, a solution containinga R-14 therapeutic can be applied as drops directly on the eye to lowerintraocular pressure.

In one embodiment, such compositions include an agent capable ofmodulating R-14 activity, such as an R-14 antagonist, in atherapeutically or prophylactically effective amount sufficient toreduce intraocular pressure, and a pharmaceutically acceptable carrier.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, monostearate salts and gelatin. Moreover, the R-14 antagonistscan be administered in a time release formulation, for example in acomposition which includes a slow release polymer. The active compoundscan be prepared with carriers that will protect the compound againstrapid release, such as a controlled release formulation, includingimplants and microencapsulated delivery systems. Biodegradable,biocompatible polymers can be used, such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylacticacid and polylactic, polyglycolic copolymers (PLG). Many methods for thepreparation of such formulations are patented or generally known tothose skilled in the art.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g. R-14 antagonist) in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclewhich contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation are vacuum drying and freeze-drying which yieldsa powder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof. In accordance withan alternative aspect of the invention, an R-14 antagonist may beformulated with one or more additional compounds that enhance thesolubility of the R-14 antagonist.

A further form of administration is to the eye. An agent or compoundcapable of modulating R-14 activity, such as an R-14 antagonist, may bedelivered in a pharmaceutically acceptable ophthalmic vehicle, such thatthe compound is maintained in contact with the ocular surface for asufficient time period to allow the compound to penetrate the cornealand internal regions of the eye, as for example the anterior chamber,posterior chamber, vitreous body, aqueous humor, vitreous humor, cornea,iris/ciliary, lens, choroid/retina and sclera. Thepharmaceutically-acceptable ophthalmic vehicle may, for example, be anointment, vegetable oil or an encapsulating material. Alternatively, thecompounds of the invention may be injected directly into the vitreousand aqueous humour. In a further alternative, the compounds may beadministered systemically, such as by intravenous infusion or injection,for treatment of the eye.

A further aspect of the present invention is a method of loweringintraocular pressure in a subject and/or preventing and/or treating acondition associated with elevated intraocular pressure (e.g. glaucoma),by administering to a subject a nucleic acid molecule encoding a peptidecompound of the invention. Suitable methods of administration includegene therapy methods.

A nucleic acid of the invention may be delivered to cells in vivo usingmethods such as direct injection of DNA, receptor-mediated DNA uptake,viral-mediated transfection or non-viral transfection and lipid basedtransfection, all of which may involve the use of gene therapy vectors.Direct injection has been used to introduce naked DNA into cells in vivo(see e.g., Acsadi et al. (1991) Nature 332:815-818; Wolff et al. (1990)Science 247:1465-1468). A delivery apparatus (e.g., a “gene gun”) forinjecting DNA into cells in vivo may be used. Such an apparatus may becommercially available (e.g., from BioRad). Naked DNA may also beintroduced into cells by complexing the DNA to a cation, such aspolylysine, which is coupled to a ligand for a cell-surface receptor(see for example Wu, G. and Wu, C. H. (1988) J. Biol. Chem. 263:14621;Wilson el al. (1992) J. Biol. Chem. 267:963-967; and U.S. Pat. No.5,166,320). Binding of the DNA-ligand complex to the receptor mayfacilitate uptake of the DNA by receptor-mediated endocytosis. ADNA-ligand complex linked to adenovirus capsids which disrupt endosomes,thereby releasing material into the cytoplasm, may be used to avoiddegradation of the complex by intracellular lysosomes (see for exampleCuriel el al. (1991) Proc. Natl. Acad. Sci. USA 88:8850; Cristiano etal. (1993) Proc. Natl. Acad. Sci. USA 90:2122-2126).

Defective retroviruses, are well characterized for use as gene therapyvectors (for a review see Miller, A. D. (1990) Blood 76:271). Protocolsfor producing recombinant retroviruses and for infecting cells in vitroor in vivo with such viruses can be found in Current Protocols inMolecular Biology, Ausubel, F. M. et al. (eds.) Greene PublishingAssociates, (1989), Sections 9.10-9.14 and other standard laboratorymanuals. Examples of suitable retroviruses include pLJ, pZIP, pWE andpEM which are well known to those skilled in the art. Examples ofsuitable packaging virus lines include .psi.Crip, .psi.Cre, .psi.2 and.psi.Am. Retroviruses have been used to introduce a variety of genesinto many different cell types, including epithelial cells, endothelialcells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitroand/or in vivo (see for example Eglitis, et al. (1985) Science230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; vanBeusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay etal. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol.150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCTApplication WO 89/07136; PCT Application WO 89/02468; PCT Application WO89/05345; and PCT Application WO 92/07573).

For use as a gene therapy vector, the genome of an adenovirus may bemanipulated so that it encodes and expresses a peptide compound of theinvention, but is inactivated in terms of its ability to replicate in anormal lytic viral life cycle. See for example Berkner et al. (1988)BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434; andRosenfeld et al. (1992) Cell 68:143-155. Suitable adenoviral vectorsderived from the adenovirus strain Ad type 5 dl324 or other strains ofadenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well known to those skilled inthe art. Recombinant adenoviruses are advantageous in that they do notrequire dividing cells to be effective gene delivery vehicles and can beused to infect a wide variety of cell types, including airway epithelium(Rosenfeld et al. (1992) cited supra), endothelial cells (Lemarchand etal. (1992) Proc. Natl. Acad. Sci. USA 89:6482-6486), hepatocytes (Herzand Gerard (1993) Proc. Natl. Acad. Sci. USA 90:2812-2816) and musclecells (Quantin el al. (1992) Proc. Natl. Acad. Sci. USA 89:2581-2584).

Adeno-associated virus (AAV) may be used as a gene therapy vector fordelivery of DNA for gene therapy purposes. AAV is a naturally occurringdefective virus that requires another virus, such as an adenovirus or aherpes virus, as a helper virus for efficient replication and aproductive life cycle (Muzyczka et al. Curr. Topics in Micro. andImmunol. (1992) 158:97-129). AAV may be used to integrate DNA intonon-dividing cells (see for example Flotte et al. (1992) Am. J. Respir.Cell. Mol. Biol. 7:349-356; Samulski et al. (1989) J. Virol.63:3822-3828; and McLaughlin et al. (1989) J. Virol. 62:1963-1973). AnAAV vector such as that described in Tratschin et al. (1985) Mol. Cell.Biol. 5:3251-3260 may be used to introduce DNA into cells (see forexample Hermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470;Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al.(1988) Mol. Endocrinol. 2:32-39; Tratschin et al. (1984) J. Virol.51:611-619; and Flotte et al. (1993) J. Biol. Chem. 268:3781-3790).Lentiviral gene therapy vectors may also be adapted for use in theinvention.

General methods for gene therapy are known in the art. See for example,U.S. Pat. No. 5,399,346 by Anderson et al. A biocompatible capsule fordelivering genetic material is described in PCT Publication WO 95/05452by Baetge et al. Methods of gene transfer into hematopoietic cells havealso previously been reported (see Clapp, D. W., et al., Blood 78:1132-1139 (1991); Anderson, Science 288:627-9 (2000); andCavazzana-Calvo et al., Science 288:669-72 (2000)).

The invention further relates to transplantation methods, to introduceinto a subject a cell comprising a nucleic acid capable of encoding apeptide compound of the invention. The nucleic acid may be present in avector as described above, the vector being introduced into the cell invitro, using for example the methods described above. In an embodiment,the cell is autologous, and is obtained from the subject. Inembodiments, the cell is allogeneic or xenogeneic.

In embodiments, the therapeutic method may be used in conjunction with adiagnostic method. For example, a subject suffering from a conditionassociated with intraocular pressure (e.g. glaucoma) may be identifiedor diagnosed using a diagnostic method and then subsequently treatedusing a therapeutic method. Further, the therapeutic method may be usedfor treatment in conjunction with the diagnostic or prognostic methodwhich is used to monitor the progress of the treatment.

In accordance with another aspect of the invention, therapeuticcompositions of the present invention, comprising a R-14 antagonist, maybe provided in containers or commercial packages which further compriseinstructions for use of the R-14 antagonist for the prevention and/ortreatment of elevated intraocular pressure and related disorders such asglaucoma.

Accordingly, the invention further provides a commercial packagecomprising an R-14 antagonist or the above-mentioned compositiontogether with instructions for the prevention and/or treatment ofelevated intraocular pressure and related disorders such as glaucoma.

The invention further provides a use of the above-noted peptides,compounds and compositions for lowering intraocular pressure in asubject and/or for the prevention and/or treatment of elevatedintraocular pressure and related disorders such as glaucoma.

The invention further provides a use of the above-noted peptides,compounds and compositions for the preparation of a medicament forlowering intraocular pressure in a subject and/or for the preventionand/or treatment of elevated intraocular pressure and related disorderssuch as glaucoma.

Although various embodiments of the invention are disclosed herein, manyadaptations and modifications may be made within the scope of theinvention in accordance with the common general knowledge of thoseskilled in this art. Such modifications include the substitution ofknown equivalents for any aspect of the invention in order to achievethe same result in substantially the same way. Numeric ranges areinclusive of the numbers defining the range. In the claims, the word“comprising” is used as an open-ended term, substantially equivalent tothe phrase “including, but not limited to”. The following examples areillustrative of various aspects of the invention, and do not limit thebroad aspects of the invention as disclosed herein.

EXAMPLES Example 1 Cloning and Characterization of the Nucleic AcidEncoding R-14

In silico analysis of EST # HTM1-025F1 (GenBank Acc.: BE439409) againsthuman genome using BLAST-N (v. 2.2.1) program resulted in a hit of 99.8%similarity to HTM1-025F1 in contig NT_(—)009307.3, AC020568.4 onchromosome 11. A 966 bp fragment was amplified from a BAC clone,RP11-206C1 obtained from the Sanger Center, UK, by PCR, using genespecific primers, 5′ GATTCAACCATCCCAGTCTTGGGTACAG 3′ (SEQ ID NO:6) and5′ TTACTGCTCCAATCTGCTTCCCGACAGC 3′ (SEQ ID NO:7). PCR was done using TaqHiFi (Invitrogen, CA) following the protocol suggested by themanufacturer (annealing at 60° C. for 30 sec, elongation at 68° C. for80 sec: total cycles—35). The PCR products were separated using 1%agarose gels. A photograph of the gel is shown in FIG. 1(A). −Bac: Noplasmid DNA; +Bac: contains plasmid DNA (0.1 μg); MW std: λ Hind IIIdigest. The ˜960 bp fragment (indicated by arrow) was cloned intopcDNA4HisMax TOPO TA (Invitrogen, CA) according to the manufacturer'sinstructions. Orientation of the cloned insert was assessed by PCR usingvector-specific and gene-specific primers (5′ TATGGCTAGCATGACTGGT 3′(SEQ ID NO:8) vector's Express epitope coding sequence and 5′TTACTGCTCCAATCTGCTTCCCGACAGC 3′ (SEQ ID NO:7) gene specific 3′ endprimer). Three clones were sequenced by the dideoxy sequencing method.

The nucleotide sequence of the R-14 reading frame is shown in FIG. 1(B).The sequences obtained from 2 clones were 99.5% homologous to HTMI-0025(Gonzalez et al., supra) except for a C (instead of an A) at position626. R14.0 protein sequence was 99.4% homologous to MGRx3, a genesequence identified during a search of mas-oncogene related sequences inmouse and human genomes (Dong X et al. [2000] Cell. 106:619).

The R-14 polypeptide sequence is shown in FIG. 1(C). The sevenunderlined sequences show potential hydrophobic transmembrane domains.Shaded residues represent potential phosphorylation sites; PKA(R-X1-2-S/T-X) or PKC (X-S/T-X-R/K).

Example 2 Expression of R-14 mRNA in Human Trabecular Meshwork Tissue

Method: RT-PCR of total mRNA isolated from human trabecular meshwork(HTM) and human iris (HI) tissues. Aliquots of total RNA (1 μg) werereverse transcribed (50° C. for 30 min) and the reaction mixture wasamplified for 35 cycles (60° C. for 30 sec) using gene-specific primersSuperScript One-Step RT-PCR (InVitrogen). Resolution of the PCR productsby agarose gel electrophoresis showed a single DNA fragment of 1 Kb fromHTM tissue, but not from iris tissue. Sequencing of the DNA fragmentfrom HTM tissue identified the 1 Kb fragment to contain the R-14 readingframe.

Example 3 Expression of Cloned Human R-14 Receptor in Human Cells

Cell culture: HEK293 cells were grown in complete DMEM (10% fetal calfserum, 0.1% Penicillin and streptomycin, 2 mM glutamine, 0.5 ug/mlfungizone, 5 ug/ml gentamicin) until ˜70% confluent. Cells weretransfected using Lipofectamine 2000 (InVitrogen) according to themanufacturer's recommendations using 12.5 μg DNA and 25 μl Lipofectaminein DMEM. Media was replaced after 48 hours for complete DMEM (The cellswere processed immediately to detect transient expression of R-14),supplemented with 500 μg/ml Zeocin. After 3 weeks in culture, thezeocin-resistant cells (R14/293) were split and cultured in completeDMEM with 100 μg/ml Zeocin.

Immunoblotting of R-14 proteins: Confluent cells were lysed in TNE (10mM Tris-HCl pH 7.4, 0.1 mM EDTA, 0.85% NaCl, 1% NP-40) buffer containingprotease inhibitor cocktail. Aliquots (100 μg protein) of supernatantswere denatured in 50 μl of SDS-loading buffer by boiling for 5 minutesand resolved on 10% SDS-acrylamide gels. Proteins were transferred toPVDF membrane by electroblotting and was blocked with 5% dry milk BLOTTOfor 1 h, and incubated with mouse anti-HIS ({fraction (1/2000)}) for 1h, washed, then incubated with goat anti mouse-HRP ({fraction (1/2000)})in TBST for 1 hour. Membrane was rinsed with TBS and developed bycolorimetry using a DAB/CN substrate (Pierce). The details ofimmunoblotting are given in Moore D et al (Ed) Current protocols inmolecular biology 1987. John Wiley & sons inc. With reference to FIG. 3,R14+ refer to HEK 293 cells expressing recombinant R14 fusion protein(HIS-tag) whereas R14− refer to parent HEK293 cells which do not containnative R-14 receptor. The immunoreactive R-14 band is indicated by anarrow.

Example 4 Identification and Characterization of Peptide Inhibitors ofR-14 Protein

Method: Newborn pigs (1-3 days old) were anesthetized with 1.5%halothane for tracheostomy and catheterization of the right femoral veinfor drug administration. Animals were ventilated by means of a Harvardsmall animal respirator with a gas mixture of 25% 02 and 75% N₂.Halothane was discontinued after surgery and immediately thereafter theanimals were sedated with Alpha-chloralose (50 mg/kg i.v.) and paralyzedwith pancuronium (0.1 mg/kg i.v.). Animals were placed under radiantwarmer to keep their body temperature at 37° C. A butterfly needle (24G)is inserted into the anterior chamber of both eyes and connected to aStatham pressure transducer connected to a Gould multichannel recorder.Intraocular pressure is then allowed to stabilize for 15 minutes.Peptides were prepared by standard chemical methods of peptidesynthesis. Peptides dissolved in saline were topically applied as dropsunder the lower eyelid and allowed to diffuse. The intraocular pressurewas monitored for 15 minutes at which time a maximal effect wasobserved.

(A) Effects of peptides on intraocular pressure in piglet. Peptidesdissolved in saline were applied to the eye after 15 min ofstabilization of intraocular pressure and changes in pressure weremonitored for 15 min. The deviation (negative for hypotension) wasplotted as a function of time (min). Of the peptides tested, 1401, 1402and 1405 produced a decrease in ocular pressure within minutes ofapplication, whereas 1406 and 1407 did not affect the basal IOP in theanimals.

(B) Dose-response of 1402 and 1405 peptides on intra-ocular pressure inpiglets. The experiments were done as above, except that intraocularpressure was plotted versus the dose of peptide used. Dose response ofthe peptides on basal IOP of piglet revealed IC50 values of 86.4 and341.6 nM for 1402 and 1405 respectively.

TABLE 4 Alternative peptides according to embodiments of the invention.Peptide SEQ ID NO: Sequence 1401 3 F S L T Q K Y C 1402 4 H I I C S P LR 1405 5 I F D S T E C W X₁ X₂ X₃ X₄ X₅ X₆ X₇ X₈

Example 5 Comparison of the Efficacy of 1405 with Latanoprost andTimolol

Method: Immediately after euthanasia, rabbit eyeballs were collected andplaced into 15-ml organ baths (Radnoti Glass, Monrovia, Calif.). Bathswere filled with Krebs (composition in mM: NaCl 120, KCl 4.5, CaCl₂ 2.5,MgSO₄ 1.0, NaHCO₃ 27, KH₂PO₄ 1.0, and glucose 10, pH 7.4), maintained at37° C. and bubbled with 95% 02. The eyeballs were maintained in placewith the cornea facing upwards. A butterfly needle (24G) is insertedinto the anterior chamber of the eye and connected to a Statham pressuretransducer connected to a Gould multichannel recorder. Intraocularpressure is then increased to 35 mmHg manually with saline. The recoveryrate (return to 20 mmHg) in the absence (control) or presence oftopically applied agents was assessed. Two measurements were averaged,both in the relatively linear portion of the recovery. Controlmeasurements were repeated twice, giving identical results. All valueswere obtained on the same rabbit eye. Ophthalmic preparations oflatanoprost (0.03%) and timolol (0.5%) in addition to peptide 1405(0.1%) in saline were applied as drops to the eyes. Peptide 1405reversed experimentally-induced ocular hypertension with an efficiencysimilar to that of timolol, but significantly faster than latanoprost.

Example 6 Selectivity of Peptides 1402 and 1405

Method: Adult pig eyecup preparations are used to study the response insitu of the relatively undisturbed retinal vasculature. Briefly, acircular incision is made 3-4 mm posterior to the ora serrata to removethe anterior segment and vitreous body with minimal handling of theretina. The remaining eyecup is fixed with pins to a wax base in a 20-mltissue bath containing Krebs buffer (pH 7.35-7.45) equilibrated with 21%02 and 5% CO₂ and maintained at 37° C. The preparations are allowed tostabilize for 30-45 min, during which they were rinsed two or threetimes with fresh buffer. Cumulative concentration-response curves todifferent agents are constructed separately on nonperfused primaryarterioles (100-200 μm diameter) of fresh tissue. The outer vesseldiameter is recorded with a video camera mounted on a dissectingmicroscope (model M-400, Zeiss), and responses are quantified by adigital image analyzer (Sigma Scan software, Jandel Scientific, CorteMadera, Calif.). Vascular diameter is recorded before and 10 min aftertopical application of each concentration of agent, at which time astable response is generally achieved. Each measurement is repeatedthree times, and variability is <1%. Additional experiments can beperformed after a 20 min pretreatment with a variety of blocking ormodulating agents. The responses are expressed as percent change in theouter diameter of vessel from baseline or as a percent reversal of aconstrictor agent (Thromboxane receptor agonist, U46619 at aconcentration producing a 70% of its maximal effects).

Both latanoprost and timolol constricted the porcine adult retinalarterioles by an average of 7-8% (FIG. 6A). The R-14 antagonist peptides1402 and 1405 were tested to measure the relative selectivity of thesecompounds compared to known ocular hypotensive compounds, latanoprostand timolol. Both 1402 and 1405 peptides did not reverse theconstriction produced by latanoprost (by 1-5%) or by timolol (<1%).

Example 7 Effect of Peptides 1402 (A) and 1405 (B) on IntraocularPressure in Rabbits

New Zealand white rabbits were trained to stay calm during theapplication of eye drops and measurement of intraocular pressure (IOP)by flourescein tonometry. Peptides were freshly made in phosphatebuffered saline at concentrations indicated and a drop (50 μl) wasapplied in the irido-corneal angle of the eye. The IOP measurements weretaken at different times. As shown in FIG. 7, there was an immediatedecrease in IOP by the application of either peptide and the IOPreturned to normal values within 4 h. Repeated application of thepeptide produced similar decreases in IOP and the effects are reversiblewith the disappearance of the peptides.

Throughout this application, various references are referred to describemore fully the state of the art to which this invention pertains. Thedisclosures of these references are hereby incorporated by referenceinto the present disclosure.

1. A substantially pure peptide compound of Formula I:Z₁-Ile-Phe-Asp-Ser-Thr-Glu-Cys-Trp-Z₂  I wherein: Z₁ is an N-terminalgroup of the formula H₂N—, RHN— or, RRN—; Z₂ is a C-terminal group ofthe formula —C(O)OH, —C(O)R, —C(O)OR, —C(O)NHR, —C(O)NRR; R at eachoccurrence is independently selected from (C₁-C₆) alkyl, (C₁-C₆)alkenyl, (C₁-C₆) alkynyl, substituted (C₁-C₆) alkyl, substituted (C₁-C₆)alkenyl, or substituted (C₁-C₆) alkynyl; and “—” is a covalent linkage.2. A substantially pure synthetic peptide compound or recombinantpeptide compound, said compound having a domain of Formula II:-Ile-Phe-Asp-Ser-Thr-Glu-Cys-Trp-  II wherein “—” is a covalent linkage.3. The compound of claim 1, wherein said compound is IFDSTECW (SEQ IDNO: 5).
 4. A method of lowering intraocular pressure in a subject, saidmethod comprising administering to said subject an effective amount ofthe compound of claim
 1. 5. A method of treating in a subject acondition associated with elevated intraocular pressure, said methodcomprising administering to said subject an effective amount of thecompound of claim
 1. 6. The method of claim 5, wherein the condition isglaucoma.
 7. The method of claim 4, wherein said method comprisesadministering said compound to an eye of said subject.
 8. The method ofclaim 4, wherein the subject is a mammal.
 9. The method of claim 8,wherein the subject is human.
 10. A pharmaceutical compositioncomprising the compound of claim 1 in admixture with a pharmaceuticallyacceptable carrier.
 11. The pharmaceutical composition of claim 10,wherein said composition is formulated for administration to an eye of asubject.
 12. A package comprising the compound of claim 1 together withinstruction for its use.
 13. The package of claim 12, wherein said useis selected from the group consisting of: (a) lowering intraocularpressure in a subject; and (b) treating a condition associated withelevated intraocular pressure.
 14. The package of claim 13, wherein saidcondition is glaucoma.
 15. A method of lowering intraocular pressure ina subject, said method comprising administering to said subject aneffective amount to the composition of claim
 10. 16. A method oftreating in a subject a condition associated with elevated intraocularpressure, said method comprising administering to said subject aneffective amount of the composition of claim
 10. 17. The method of claim16, wherein the condition is glaucoma.
 18. The method of claim 15,wherein said method comprises administering said composition to an eyeof said subject.
 19. The method of claim 15, wherein the subject is amammal.
 20. The method of claim 19, wherein the subject is human.
 21. Apackage comprising the composition of claim 10, together withinstructions for its use.
 22. The package of claim 21, wherein said useis selected from the group consisting of: (a) lowering intraocularpressure in a subject; and (b) treating a condition associated withelevated intraocular pressure.
 23. The package of claim 22, wherein saidcondition is glaucoma.
 24. A method of lowering intraocular pressure ina subject, said method comprising administering to said subject aneffective amount of the compound of claim
 2. 25. A method of treating ina subject a condition associated with elevated intraocular pressure,said method comprising administering to said subject an effective amountof the compound of claim
 2. 26. The method of claim 25, wherein thecondition is glaucoma.
 27. The method of claim 24, wherein said methodcomprises administering said compound to an eye of said subject.
 28. Themethod of claim 24, wherein the subject in a mammal.
 29. The method ofclaim 28, wherein the subject is human.